Abstract We present the introduction of a new generation of techniques for early and specific diagnosis of inflammatory, immune and degenerative eye diseases. We concentrate on the applications of the SELDI-TOF (Surface enhanced laser desorption ionisation - time of flight) mass spectroscopy in combination with molecular modelling and visualization for the analysis of the aqueous humour. For our study we used aqueous humour from 25 patients undergoing cataract operation. SELDI-TOF consists on one part of an apparatus allowing the laser bombarding of molecules on a solid surface and on the other part on a system which measure the time of flight of the molecules. During the measurement the molecules are liberated from the matrix and carried to the detector. Specific proteins can be identified by the characteristic peaks of the spectrogram. Albumin and gamma-globulin were found to a considerable extend in the aqueous humour. An increased amount has been detected in inflammatory humour. The quantity may reflect some underlying complications such as inflammation and animate for further research on the spectrum of proteins and peptides released by underlying eye disorder. SELDI-TOF delivers characteristic peptide mass fingerprints, which can be used as input for the prediction of the tertiary protein structure with homology modelling. Structural deviations between a genuine protein and a mutated can be an indicator about the underlying disease, paving the way for successful diagnostic and therapy. The combination of two new technologies namely SELDI-TOF and molecular modelling may offer a broad spectrum for the differential diagnosis of some eye diseases.

Abstract Endothelial vasodilator dysfunction is a characteristic feature of patients at risk for coronary atheroscierosis. We have reported that insulin resistance may be a pathogenic factor for endothehal dysfunction through impaired endothelial nitric oxide synthase (eNOS) activity and increased oxidative breakdown of nitric oxide (NO) due to an enhanced fonnation of Superoxide anion, which arc caused by relative deficiency of tetrahydrobiopterin (BH 4 ) in vascular endothelial cclls. Guanosine-triphosphate cyclohydrolase I, the rate-limiting cnzyme in the production of BH 4 , is decreased in the aorta of insulin-resistant rats and supplementation of BH 4 restored the endothelial function and relieved oxidative tissue damage. The BH 4 treatment may evoke these benefits not only by providing eNOS with cofactor to enhance NO synthesis, but also by acting as an indirect and/or direct antioxidant to decrease Superoxide anion derived from the endothelium. A further understanding of the physiological and pathological roles and their regulation may lead to new therapeutic avenues.

Abstract Two types of pteridine - polyamine conjugates designed on oncopterin perform different actions to DNA. The most obvious difference is their actions in DNA compaction. One of the pteridine - polyamine conjugates, PT- 2R, containing a polyamine substituent (R) on the C(2) position of pterin (PT) compacts a DNA molcculc in an all-or-none fashion without the intervention of an intermediate. The othcr (PT-4R: R exists on the c(4) position of PT) carries out the DNA compaction via partially compacted intermediates. The difference is discussed based on various interaction modes of the pteridine - polyamine conjugates to DNA.

Abstract Extract of traditional Tibetan herbal remedy Padma 28 revealed suppressive effects on neopterin production and tryptophan degradation in mitogen-stimulated peripheral blood mononuclear cells (PBMC). One of the questions was to asses the robustness of the Tibetan way of formulating herbal multicomponent medicines. Can small changes affect the effectiveness of the formula? In this study, effect of a derivative multicomponent mixture was investigated in stimulated and unstimulated human PBMC in vitro. Neopterin production and tryptophan degradation were measured in supernatants of PBMC in the presence or absence of mitogens phytohacmagglutinin and concanavalin A. Stimulation of PBMC induced neopterin formation and tryptophan degradation, and the herbal multicomponent mixture inhibited both immunobiochemical effects in a dose-dependent way. Higher concentrations were more effective and were able to completely block the pathways induccd upon mitogenic stimulation. When comparing the results obtained with those observed earlier using Padma 28, there was no obvious difference between the two different preparations. Data allow to conclude that Padma 28 and its modified version is equally able to suppress immunobiological effects in stimulated PBMC.

Abstract Patients with AD often show altered levels of some immune molecules in their peripheral blood which correlate with cognitive impairment. Downs' syndrome (DS) subjects are at high risk of developing Alzheimer's disease (AD). Here we studied immune molecules, such as interleukin-6 (IL-6), C reactive protein (CRP) and neopterin, in the blood of non demented children with DS to investigate whether altered peripheral immune phenotype could be present in this subjects without dementia, many years before the presentation of clinical signs of cognitive deterioration. Plasma levels of neopterin and IL-6 were measured by commercially available ELISA kits, whereas plasma CRP concentration was evaluated by nephelometric immunoassay technique. We studied a group of 40 patients with DS, 20 healthy controls and 100 patients w ith AD. Plasma levels of IL-6 and CRP were significantly higher in DS than in control children. The increase of IL-6 and CRP from DS children was similar to that found in elderly patients with clinical AD. In the present research we have also studied blood levels of neopterin which were in the normal range either in DS or control. Increased or normal levels of this metabolite have been found in the blood of AD patients. However, increased blood neopterin was likely to be only elevated in patients with advanced clinical stage of AD. Peripheral altered immune phenotype in healthy young subjects with DS might be an early sign of CNS alterations leading many years later to cognitive deterioration and dementia.

Abstract Assessment of markers of systemic inflammation, such as acute-phase reactants C-reactive protein (CRP), fibrinogen and ceruloplasmin, sialic acid, a major component of these proteins and neopterin, a specific marker of cellular immune activation, in clinically stable coronary artery disease, may contribute to Staging and risk stratification. These markers were measured in 225 consecutive stable coronary artery disease patients before undergoing coronary angiography. According to their angiographic scores 32 patients were designated as having minor, 34 moderate and 96 severe coronary artery disease. 63 patients with negative angiograms were taken as the angiographic controls. High sensitive-CRP (hs-CRP) and fibrinogen were found to be higher in patients with angiographically established coronary artery disease, than in angiographic controls (p<0.05) and correlated with the severity and extent of disease. Ceruloplasmin and sialic acid concentrations did not differ between patients with and without angiographically cstablished coronary artery disease. Serum neopterin levels were sigificantly higher in patients undergoing coronary angiography than in healthy controls. Neopterin levels were similar between the different subgroups of coronary artery disease, suggesting that neopterin determinations do not contribute to the assessment of the presence and severity of disease in clinically stable patients. In stable angina, serum hs-CRP and plasma fibrinogen levels proved to be more effective than ceruloplasmin, sialic acid and neopterin in discriminating between patients with positive and negative angiograms and various degrees of coronary artery disease, thus in pointing out to increased risk. Our results, do not support the inclusion of neopterin in risk assessment of stable coronary artery disease.

Abstract Neopterin is released from human monocyte-derived macrophages preferentially upon stimulation with the Thlcell cytokine interferon-γ. In humans, increased concentrations of neopterin in serum and urine have been found in viral infections including human immunodeficiency virus type 1, various malignant disorders, autoimmune diseases and during allograft rejection episodes. In order to find additional parameters which might influence neopterin production, serum neopterin concentrations of 1156 blood donors were compared with other parameters routinely determined in blood transfusion. There existed correlations between serum neopterin concentration and blood donors' age (Spearman's rank correlation: r s = 0.259, ρ <0.0001), arterial diastolic blood pressure (r s 0.132, ρ <0.0001) and body mass index (r s = 0.084, ρ <0.01). Serum neopterin concentrations were lower in smokers compared to nonsmokers (Mann-Whitney test, ρ <0.0001). The data demonstrate that higher neopterin concentrations are associated with older age, higher diastolic blood pressure and higher body mass index, whereas lower neopterin concentrations are found in smokers compared to nonsmokers. It is assumed that immunopathogenetic pathways underlie these relationships, but the exact background of these associations still needs to be resolved.

Abstract 1,3-Dimethyllumazine-6,7-diamine (1) and its monomethyl -(3,4) and monophenylamino (5) derivates are interesting starting materials for the synthesis of imidazo[4,5-g] (6-22) and pyrazino[2,3-g]lumazines (23-26). Ringclosure proceeds under relative drastic conditions and the resulting reaction products are highly fluorescent. Methylation of 5,7-dimethyll-imidazo[4,5-g)lumazine (6) led to an isomeric mixture of the 1- (17) and 3-methyl derivatives (7) which could be separated chromatographically. Their structural assignment was based on unambiguous syntheses heating 4 and 3, respectively, with formamide under reflux for ringclosure. The newly synthesized compounds have been characterized by 1 H-NMR and UV-spectra as well as elemental analyses.

Abstract Three exact criteria are first derived for the probabilities that determine single molecule sensitivity in single-phases, for example in solutions or membranes. It is shown how the criteria can be used to decide whether single molecule sensitivity is obtained. Most straightforwardly, all we require is that we experimentally determine the Poisson probability for the absolute number of fluorescent molecules in the volume of observation. This is achieved by fluorescence correlation spectroscopy and allows identifying the selfsame, one single fluorescent molecule in single-phases. Further, we first provide the new and powerful analytical capability to fluorescencetagged inidividual molecules and reactions in in-vitro and living systems to follow their molecular interactions and function by means of spatial stochastic behavior. This fully stochastic modeling is derived which describes the influence of spatial separation on the reaction coordinate of one single individual molecule in single-phases within the observation volume. The theoretical concept is applied to experimental data 'at the single-molecule level' of photosensitive riboflavin and riboflavin-containing blue fluorescence protein. The data were taken from the literature. As simulation results of the elucidative examples, experiments are suggested that would measure individual, single molecules of flavins and flavoproteins m solution. They are of importance for experimentalists.