Volume 12, Issue 4

The in vitro response of early haematopoietic progenitors or stem cells, common for myeloid (granulocyte, eosinophil, megacaryocyte) and marrow stromal (including dendritic CD34+) cells series, to neopterin, exogenously added to the liquid- and semi-liquid (agar-) mouse bone marrow cultures, at doses 12.5 - 25 Jlg/ml culture medium, has been studied. The results obtained show a significant stimulation of common myeloid and stromal dendritic cell (CD34+) progenitors' proliferation and/or differentiation as early as 24h after the in vitro treatment of marrow cultures with neopterin. On day 4 of cultivation the granulocyte/macrophageal proliferation and differentiation has been attenuated giving place to the marrow stromal dendritic cell (probably CD34+) differentiation. The engagement of the nuclear proliferative factor NF-K B may playa role in the light of recent data that neopterin could activate this transcriptionally active nuclear factor. The significance of clonal selective stimulation of healthy (but not of leukaemic) dendritic CD34+ cells in some pathological cases as acute myeloid leukaemia has been also discussed because neopterin could be a more efficient marrow stem cell factor than cytokine cocktails with granulocyte/macrophage-colony stimulating factor. The ex vivo or clinical application of neopterin, alone and/or in specific combinations with other cytokines, in the induction of marrow myeloid and stromal cell proliferation and differentiation towards the dendritic (CD34+) cells merits further investigations ..

In a Number of malignant diseases including ovarian carcinomas, increased serum or urine levels of neopterin serve as a valuable marker for the state of activation of the cellular immune system. Reported correlations benveen elevated neopterin concentrations and shorter survival time of the tumor host might be explained by direct biochemical actions exhibited by the pteridine compound. In the present study, we investigated the influence of neopterin on inducible nitric oxide synthase (iNOS) gene expression in two ovarian carcinoma cell lines. Qualitative and quantitative analyses of iNOS as well as measurements of nitric oxide (NO) metabolites in the cell supernatant revealed an inhibitory effect of neopterin (1000 11M) on NO generation induced by a combination ofinterferon-γ (100 U/ml), tumor necrosis factor-a (500 U/ml), and interleukin-1β (10 U/ml). Since NO is most likely involved in apoptotic cell death in ovarian carcinoma cells, it is conceivable that suppression of NO synthesis represents a biochemical mechanism mediated by neopterin that is advantageous to tumor progression and thus harmful to the host.

In the present investigation, fluorescence con-elation spectroscopy (FCS) was used to measure the molecular motion of the pteridine derivative neopterin. However, technical limitations in the present optical setup precluded the identification of ,single neopterin molecules. FCS measurements with a fluorophore were also can-ied out for comparison. Exemplified by rhodamine green, we have introduced a concept that allows the detection, identification and analysis of assays in solution at the single-molecule level in tenns of bulk concentration. This concept is based on FCS and Poisson distribution analysis of assay sensitivity. The molecules had not to be quantified in a more concentrated fonn, or in flow and trapping experiments. The study demonstrated an ultrasensitive, reliable, rapid and direct tool for analytics and diagnostics in solution. We discuss a possible application of our new concept in activation control of cell-mediated immunity via neopterin determination.

Increased production of neopterin in humans is indicative for an activated cellular immune response. The intestinal immune system encounters an enormous amount of infectious agents or injurious substances evoking a cellular immune response. The objective of this pilot study was to measure neopterin levels in colon dialysates of healthy individuals and in comparison with individuals under a starvation diet and with patients. In parallel, urinary and salivary neopterin levels were determined in these subjects. The mean neopterin concentration in colon dialysate was about J 0, times higher than in serum. These high neopterin values agree with a great impact of the cellular immune system within the human colon. Common diseases had a strong and prompt effect on the neopterin levels in colon dialysate, e.g. three individuals with acute gastritis had very high neopterin concentrations. In subjects with a starvation diet according to F.X.Mayr, which is characterized by low fiber and low calorie intake, the neopterin levels in colon dialysate were lower. The data may argue for a regeneration of the cellular immune system during fasting.

We examined the role ofprotein kinase C (PKC) in the insulin-induced biosynthesis oftetrahydrobiopterin (BH4)' which is one of the co factors of nitric oxide (NO) synthase (NOS), in vascular endothelial cells. The BH. level was determined as biopterin by reversed-phase high performance liquid chromatography with fluorometric detection. Measurement of the level ofrnRNA for GTP cyclohydrolase I (GTPCH), which is the fust and the rate-limiting enzyme for the novo pathway for BH4 synthesis, was performed by reverse transcription-polymerase chain reaction (RT-PCR). Treatment with insulin increased the BH4 level and the GTPCH rnRNA level in endothelial cells. The insulin-induced increases of both GTPCH rnRNA and BH4 levels were enhanced by co-treatment with bisindolylmaleimide I or Ro 31-8220, inhibitors of PKC. On the other hand, the insulin-induced increases of BH4 synthesis and expression of GTPCH rnRNA were also enhanced by co-incubation with a PKC activator, phorbol 12-myristate 13-acetate (PMA) for 24 h. Long-term treatment with PMA is known to down-regulate PKC. Addition of insulin after pretreatment with PMA for 24 h markedly enhanced BH4 synthesis and expression of .:JTPCH rnRNA. These findings suggest that PKC negatively regulates the induction of BH4 synthesis by insulin -:ndothelial cells. Insulin-induced BH4 synthesis may be suppressed in diabetes mellitus, since PKC has been shown to be up-regulated in diabetes mellitus.

The first human phaeochromocytorna cell line KNA was tested with regard to stimulation of dopamine biosynthesis by nicotinamide adenine dinucleotide (NADH). Differently from rat phaeochromocytoma cells - clone PC12, where NADH increased dopamine biosynthesis significantly, no stimulation in KNA cells was found. NADH is effective on speeding up the formation of the active cofactor tetrahydrobiopterin by a recycling pathway via the dihydropteridinreductase system. Another way of formation of tetrahydrobiopterin is the induction of de novo synthesis by cytokines such as interferon-y or by lipopolysaccharide via activation of GTP cyclohydrolase I, as shown in rnacrophages. Neither rat PC12 nor human KNA cells showed in vitro a stimulation of tetrahydrobiopterin de novo synthesis by interferon-y and/or lipopolysaccharide.

Folate profiles and DNA synthesis were studied during the early germinating period in Vigna radiata. Total folate andmethyl folate forms were increased from 4 to 32 hours during germination period studied. Formyl folates, involved in purine synthesis, were raised 30-fold after 26 hours of incubation followed by DNA synthesis, indicatmg that folate synthesis precedes nucleotide synthesis channelising the one-carbon moieties into nucleotide precursors. This is also supported by the observation of folate mediated one-carbon pool incorporation of 2-'4C-Histidine into nucleic acid during this period. The two folate enzymes, folylpolyglutamate hydrolase which was greatly lowered and folylpolyglutamate synthetase which was significantly elevated, might playa crucial role regulating a balanced supply of the required folate precursors during the early germination period of Vigna radiata.