PubMed Health⌕ Search

Biomedical subjects

G Werner-Felmayer

Publications and source records attributed to G Werner-Felmayer.

At least 55 records · Page 3Linked to original sources
PubMed1994

Pteridine biosynthesis and nitric oxide synthase in Physarum polycephalum.

Physarum polycephalum, an acellular slime mould, serves as a model system to study cell-cycle-dependent events since nuclear division is naturally synchronous. This organism was shown to release isoxanthopterin which is structurally related to tetrahydrobiopterin, a cofactor of aromatic amino acid hydroxylases and of nitric oxide synthases (NOSs) (EC 1.14.13.39). Here, we studied Physarum pteridine biosynthesis in more detail and found that high amounts of tetrahydrobiopterin are produced and NOS activity is expressed. Physarum pteridine biosynthesis is peculiar in as much as 7,8-dihydroneopterin aldolase (EC 4.1.2.25), an enzyme of folic acid biosynthesis usually not found in organisms producing tetrahydrobiopterin, is detected in parallel. NOS purified from Physarum depends on NADPH, tetrahydrobiopterin and flavins. Enzyme activity is independent of exogenous Ca2+ and is inhibited by arginine analogues. The purified enzyme (with a molecular mass of 130 kDa) contains tightly bound tetrahydrobiopterin and flavins. During the synchronous cell cycle of Physarum, pteridine biosynthesis increases during S-phase whereas NOS activity peaks during mitosis, drops at telophase and peaks again during early S-phase. Our results characterize Physarum pteridine biosynthesis and NOS and suggest a possible link between NOS activity and mitosis.

Amino Acid Oxidoreductases↗
PubMed1994

Synthesis and characterization of 3H-labelled tetrahydrobiopterin.

We synthesized [3'-3H]-5,6,7,8-tetrahydrobiopterin from [8,5'-3H]guanosine 5'-triphosphate ([8,5'-3H]GTP) using GTP cyclohydrolase (EC 3.5.4.16), 6-pyruvoyltetrahydropterin synthase and sepiapterin reductase (EC 1.1.1.153). After purification by cation-exchange h.p.l.c. a solution of radiochemically pure (> 95%) [3'-3H]-5,6,7,8-tetrahydrobiopterin with a specific activity of 9.2 Ci/mmol was obtained. The product proved well suited for studying the binding of tetrahydrobiopterin to nitric-oxide synthase.

Amino Acid Oxidoreductases↗
PubMed1994

Neopterin derivatives together with cyclic guanosine monophosphate induce c-fos gene expression.

We have previously shown that neopterin enhances hydrogen peroxide and chloramine T activity in a luminol-dependent chemiluminescence assay and strengthens toxicity of these agents against bacteria at slightly alkaline pH (pH 7.5), while 7,8-dihydroneopterin was shown to be a scavenger independent of the pH value. Besides various oxidants, phenolic antioxidants were shown to specifically induce expression of the c-fos and c-jun mRNAs. Using an inducible cfosCAT reporter transactivation system we studied the function of the pteridine derivatives on c-fos transactivation. For the first time, we demonstrate that neopterin and 7,8-dihydroneopterin, particularly together with cyclic guanosine monophosphate, induce c-fos gene expression. In humans, interferon-gamma induces the release of neopterin and 7,8-dihydroneopterin and also the synthesis of nitric oxide radical which in turn stimulate the formation of cGMP. Thus, in certain situations all three substances, namely neopterin, 7,8-dihydroneopterin and cGMP, may be present locally and even in the circulation at the same time. Based on our findings this constellation would significantly enhance the risk of c-fos gene expression and therefore promote tumour growth and development.

3T3 Cells↗
PubMed1994

Iron regulates nitric oxide synthase activity by controlling nuclear transcription.

Recently, it was reported that nitric oxide (NO) directly controls intracellular iron metabolism by activating iron regulatory protein (IRP), a cytoplasmic protein that regulates ferritin translation. To determine whether intracellular iron levels themselves affect NO synthase (NOS), we studied the effect of iron on cytokine-inducible NOS activity and mRNA expression in the murine macrophage cell line J774A.1. We show here that NOS activity is decreased by about 50% in homogenates obtained from cells treated with interferon gamma plus lipopolysaccharide (IFN-gamma/LPS) in the presence of 50 microM ferric iron [Fe(3+)] as compared with extracts from cells treated with IFN-gamma/LPS alone. Conversely, addition of the iron chelator desferrioxamine (100 microM) at the time of stimulation with IFN-gamma/LPS increases NOS activity up to 2.5-fold in J774 cells. These effects of changing the cellular iron state cannot be attributed to a general alteration of the IFN-gamma/LPS signal, since IFN-gamma/LPS-mediated major histocompatibility complex class II antigen expression is unaffected. Furthermore, neither was the intracellular availability of the NOS cofactor tetrahydrobiopterin altered by treatment with Fe(3+) or desferrioxamine, nor do these compounds interfere with the activity of the hemoprotein NOS in vitro. We demonstrate that the mRNA levels for NOS are profoundly increased by treatment with desferrioxamine and reduced by Fe(3+). The half-life of NOS mRNA appeared not to be significantly altered by administration of ferric ion, and NOS mRNA stability was only slightly prolonged by desferrioxamine treatment. Nuclear run-off experiments demonstrate that nuclear transcription of cytokine-inducible NOS mRNA is strongly increased by desferrioxamine whereas it is decreased by Fe(3+). Thus, this transcriptional response appears to account quantitatively for the changes in enzyme activity. Our results suggest the existence of a regulatory loop between iron metabolism and the NO/NOS pathway.

Amino Acid Oxidoreductases↗
PubMed1994

Enhancement of hydrogen peroxide-induced luminol-dependent chemiluminescence by neopterin depends on the presence of iron chelator complexes.

We have previously shown that neopterin, 6-D-erythro-trihydroxypropyl-pteridine, synthesized by human monocytes/macrophages upon stimulation by interferon-gamma, enhances toxicity of reactive oxygen at neutral or slightly alkaline pH (7.5), but not at acidic pH (below 6.5). In the present study, we explored in more detail the necessary requirements for neopterin to modulate the effects of hydrogen peroxide in a luminol-dependent chemiluminescence assay. We demonstrate that neopterin enhances hydrogen peroxide effects only in the presence of iron chelator complexes like iron-(III)- or iron-(II)-EDTA or iron-(III)-DTPA. Thus, iron chelator complexes together with neopterin may play an important role in macrophage-mediated effector mechanisms.

Biopterins↗
PubMed1994

Coinduction of nitric oxide synthesis and intracellular nonheme iron-nitrosyl complexes in murine cytokine-treated fibroblasts.

Murine fibroblasts treated with interferon-gamma plus tumor necrosis factor-alpha plus lipopolysaccharide produce nitrite and EPR-observable intracellular nonheme iron-thiol-dinitrosyl species. Inhibition of .NO synthesis or de novo tetrahydrobiopterin (BH4) synthesis decreases nitrite and the EPR signal. The effects of BH4 synthesis inhibition are prevented by sepapterin, which increases BH4 through the salvage pathway.

Animals↗
PubMed1993

Induction of GTP cyclohydrolase I by bacterial lipopolysaccharide in the rat.

A 2- to 3-fold increase of GTP cyclohydrolase I (E.C. 3.5.4.16), the key enzyme of tetrahydrobiopterin biosynthesis from GTP, was observed in cerebellum, remaining brain, liver, spleen, and adrenal gland of rats treated with a single dose of lipopolysaccharide (LPS). This led to increased biopterin levels in tissues but not in plasma. Parallel induction of nitric oxide (NO) synthase was indicated by a 10- to 100-fold increase of plasma nitrate levels 6 and 12 hours after injection of LPS. Furthermore, systolic blood pressure was reduced significantly by 23%. Our results demonstrate induction of tetrahydrobiopterin biosynthesis after LPS treatment in vivo.

Animals↗
PubMed1993

Neopterin modulates toxicity mediated by reactive oxygen and chloride species.

Neopterin, a pyrazino-pyrimidine derivative, is synthesized in excess by human monocytes/macrophages upon stimulation with interferon-gamma, a cytokine derived from activated I cells. Neopterin is furthermore produced constitutively. A relatively constant ratio between neopterin and its reduced form. 7,8-dihydroneopterin, has been described in human serum. In the study presented here we tested the ability of neopterin and its reduced form to modulate the effects of cytotoxic substances like hydrogen peroxide or hypochlorous acid and N-chloramine derivatives. We show that 7,8-dihydroneopterin potently reduces biological and chemical effects of these substances independently from the pH value. In contrast, at slightly alkaline pH (pH 7.5) neopterin enhances hydrogen peroxide and chloramine-T activity. This is demonstrated by increase of signal intensity in a luminol assay and also by enhancement of toxicity towards bacteria. Thus, the macrophage derived substance neopterin is able both to enhance and to reduce cytotoxicity in dependence of pH value and its oxidation state, and it may have a pivotal role in modulation of macrophage mediated effector mechanism.

Animals↗
PubMed1993

Pteridine biosynthesis in human endothelial cells. Impact on nitric oxide-mediated formation of cyclic GMP.

Stimulation of nitric oxide (NO) synthase in endothelial cells by Ca2+ influx leads to increased intracellular levels of cGMP. NO synthase from various sources is known to use tetrahydrobiopterin, flavins, and NADPH as cofactors. We studied the effect of interferon-gamma, tumor necrosis factor-alpha, and lipopolysaccharide on tetrahydrobiopterin biosynthetic activities in human umbilical vein endothelial cells (HUVEC). These stimuli led to an up to 40-fold increase of GTP cyclohydrolase I (EC 3.5.4.16) activity and to increased accumulation of neopterin and tetrahydrobiopterin in HUVEC. Further enzyme activities of tetrahydrobiopterin biosynthesis, i.e. 6-pyruvoyl tetrahydropterin synthase and sepiapterin reductase (EC 1.1.1.153), remained unchanged. NO synthase activity in protein fractions from homogenates of cells treated with interferon-gamma plus tumor necrosis factor-alpha was not influenced as compared with untreated controls. However, interferon-gamma alone or in combination with tumor necrosis factor-alpha significantly increased intracellular cGMP formation in intact HUVEC by 50 and 80%, respectively. These stimuli increased intracellular tetrahydrobiopterin concentrations up to 14-fold. NO-triggered cGMP formation was similarly increased by incubation of otherwise untreated cells with sepiapterin, leading to elevated intracellular tetrahydrobiopterin levels. Thus, cytokines indirectly stimulate the activity of constitutive NO synthase in HUVEC by upregulating production of the cofactor tetrahydrobiopterin.

Amino Acid Oxidoreductases↗
PubMed1993

Ca2+/calmodulin-dependent nitric oxide synthase activity in the human cervix carcinoma cell line ME-180.

We show here that the human cervix carcinoma cell line ME-180 expresses a constitutive nitric oxide (NO) synthase, as demonstrated by formation of [3H]citrulline and nitrite. The enzyme is dependent on tetrahydrobiopterin, NADPH, flavins and Ca2+/calmodulin. Enzyme activity is located in the cytosol rather than in the membrane fraction and can be inhibited by NG-monomethyl-L-arginine (NMMA). An antiserum to NO synthase purified from porcine cerebellum inhibited the enzyme activity. ME-180 cells released NO, as was shown by stimulation of guanylate cyclase (EC 4.6.1.2) in RFL-6 detector cells; this release was stimulated 8-fold by the Ca2+ ionophore A23187 and 2-fold by increasing the intracellular tetrahydrobiopterin levels with cytokines. This is the first characterization of a Ca2+/calmodulin-dependent NO synthase activity in human epithelial-type tumour cells.

Amino Acid Oxidoreductases↗
PubMed1993

Translational regulation via iron-responsive elements by the nitric oxide/NO-synthase pathway.

Nitric oxide (NO) produced from L-arginine by NO synthases (NOS) is a transmitter known to be involved in diverse biological processes, including immunomodulation, neurotransmission and blood vessel dilatation. We describe a novel role of NO as a signaling molecule in post-transcriptional gene regulation. We demonstrate that induction of NOS in macrophage and non-macrophage cell lines activates RNA binding by iron regulatory factor (IRFs), the central trans regulator of mRNAs involved in cellular iron metabolism. NO-induced binding of IRF to iron-responsive elements (IRE) specifically represses the translation of transfected IRE-containing indicator mRNAs as well as the biosynthesis of the cellular iron storage protein ferritin. These findings define a new biological function of NO and identify a regulatory connection between the NO/NOS pathway and cellular iron metabolism.

Amino Acid Oxidoreductases↗
PubMed1993

A note on the low-dimensional display of multivariate data using neural networks.

A novel neural network technique has been proposed (Livingstone et al. J. Mol. Graphics 1991, 9, 115-118) which is useful for a low-dimensional display of multivariate data sets. The method makes use of the activity values of the hidden neurons in a trained three-layer feed-forward network to produce the low-dimensional display. It was claimed that in contrast to conventional techniques, such as principal components analysis or nonlinear mapping, this technique could be used also to reconstruct, from a given point in the low-dimensional display, the corresponding multivariate input vector via the completely known weight matrices of a suitably trained network. We show here that this claim is unjustified in this general form. When previously unknown, grossly different input vectors are presented to the trained network, they can occupy, for example, exactly the same point in the low-dimensional display which is occupied also by a given training vector, if certain linear relationships between the vector components are fulfilled. Thus, an infinite set of different linearly dependent input vectors is projected onto one single point in the low-dimensional display. Reconstruction of a multivariate vector, starting from this point in the low-dimensional display, is able to lead back to only one multivariate vector (in the example given, to the original training vector).

Guanine↗
PubMed1993

Comparative effects of heme and metalloporphyrins on interferon-gamma-mediated pathways in monocytic cells (THP-1).

Previous results have demonstrated links between cell-mediated immunity, interferon (IFN)-gamma and neopterin production with heme, porphyrins, and iron metabolism. In this study, we compared the effects of heme, several metalloporphyrins, protoporphyrin IX, and iron on the signal or IFN-gamma-mediated pathways, such as the expression of major histocompatibility complex class II antigens, neopterin formation, and the degradation of tryptophan. Using the human monocytic cell line, THP-1, we found that heme, Zn-mesoporphyrin, Zn-deuteroporphyrin, Co-protoporphyrin, and iron reduced the efficiency of the IFN-gamma signal. In addition, Zn-mesoporphyrin almost fully inhibited IFN-gamma-induced degradation of tryptophan by the heme protein, indoleamine 2,3-dioxygenase. In contrast, tin-protoporphyrin enhanced the IFN-gamma effects as seen by increased neopterin production, enhanced tryptophan degradation, and elevated HLA-DR antigen expression on cells. These effects are considered to be due to the action of heme, metalloporphyrins, iron, or heme byproducts on the IFN-gamma signal, rather than to direct effects on IFN-gamma-induced enzymatic pathways. Heme and metalloporphyrins were previously shown to affect heme oxygenase activity, T cell growth, and lipid peroxidation and to modulate interleukin 2 activity. These pathways are also known to be influenced by IFN-gamma, and our data suggest that heme and metalloporphyrins may directly modulate the efficiency of the IFN-gamma signal.

Biopterins↗