The Drosophila functional Smad suppressing element fuss, a homologue of the human Skor genes, retains pro-oncogenic properties of the Ski/Sno family.

Over the years Ski and Sno have been found to be involved in cancer progression e.g. in oesophageal squamous cell carcinoma, melanoma, oestrogen receptor-positive breast carcinoma, colorectal carcinoma, and leukaemia. Often, their prooncogenic features have been linked to their ability of inhibiting the anti-proliferative action of TGF-ß signalling. Recently, not only pro-oncogenic but also anti-oncogenic functions of Ski/Sno proteins have been revealed. Besides Ski and Sno, which are ubiquitously expressed other members of Ski/Sno proteins exist which show highly specific neuronal expression, the SKI Family Transcriptional Corepressors (Skor). Among others Skor1 and Skor2 are involved in the development of Purkinje neurons and a mutation of Skor1 has been found to be associated with restless legs syndrome. But neither Skor1 nor Skor2 have been reported to be involved in cancer progression. Using overexpression studies in the Drosophila eye imaginal disc, we analysed if the Drosophila Skor homologue Fuss has retained the potential to inhibit differentiation and induce increased proliferation. Fuss expressed in cells posterior to the morphogenetic furrow, impairs photoreceptor axon pathfinding and inhibits differentiation of accessory cells. However, if its expression is induced prior to eye differentiation, Fuss might inhibit the differentiating function of Dpp signalling and might maintain proliferative action of Wg signalling, which is reminiscent of the Ski/Sno protein function in cancer.

Elevated plasma human urotensin-II-like immunoreactivity in ischemic cardiomyopathy.

BACKGROUND: The recently discovered, vasoactive, cyclic undecapeptide human urotensin-II (hU-II), and its G-protein coupled receptor (GPR14) are both expressed in the human cardiovascular system. Little is known about the pathophysiological relevance of hU-II. We hypothesised that circulating hU-II is elevated in patients with coronary artery disease (CAD) corresponding to the degree of cardiac dysfunction. METHODS: 38 patients were diagnosed with coronary artery disease by left heart catheterization, and their functional status was classified according to the New York Heart Association (NYHA). hU-II-like immunoreactivity (hU-II-LI) was measured using a novel specific and sensitive enzyme-linked immunoassay. Calculations were performed with log-transformed hU-II-LI values. RESULTS: hU-II-LI correlated positively with left ventricular end diastolic pressure (LVEDP) (r=0.32, P=0.05) and tended to correlate inversely with left ventricular ejection fraction (LV-EF) (r=-0.31, P=0.061). There was a positive correlation between hU-II-LI and NYHA class (r=0.53, P=0.001). Circulating hU-II-LI was significantly higher in patients with NYHA class III (4822+/-723 pg/ml, N=13) than in patients with class I (1884+/-642 pg/ml, N=9, P=0.007) or class II (2294+/-426 pg/ml, N=15, P=0.046). There was no difference between classes I and II (P=0.83). Furthermore, hU-II-LI correlated significantly with B-type natriuretic peptide, a marker for heart failure (r=0.40, P=0.025). In a linear regression analysis, NYHA class was the only significant independent predictor of hU-II-LI. CONCLUSIONS: The present study demonstrates that plasma hU-II-LI rises significantly in proportion to parameters of cardiac dysfunction and functional impairment in patients with coronary artery disease. These results suggest a pathophysiological role for hU-II in cardiac disease and warrant further investigation.

Identification of residues crucially involved in the binding of the heme moiety of soluble guanylate cyclase.

Soluble guanylate cyclase (sGC), a heterodimeric hemeprotein, is the only receptor for the biological messenger nitric oxide (NO) identified to date and is intimately involved in various signal transduction pathways. By using the recently discovered NO- and heme-independent sGC activator BAY 58-2667 and a novel cGMP reporter cell, we could distinguish between heme-containing and heme-free sGC in an intact cellular system. Using these novel tools, we identified the invariant amino acids tyrosine 135 and arginine 139 of the beta(1)-subunit as crucially important for both the binding of the heme moiety and the activation of sGC by BAY 58-2667. The heme is displaced by BAY 58-2667 due to a competition between the carboxylic groups of this compound and the heme propionic acids for the identified residues tyrosine 135 and arginine 139. This displacement results in the release of the axial heme ligand histidine 105 and to the observed activation of sGC. Based on these findings we postulate a signal transmission triad composed of histidine 105, tyrosine 135, and arginine 139 responsible for the enzyme activation by this compound and probably also for transducing changes in heme status and porphyrin geometry upon NO binding into alterations of sGC catalytic activity.

Preparation of heme-free soluble guanylate cyclase.

Soluble guanylate cyclase (sGC), a heterodimer consisting of alpha- and beta-subunit, is the key enzyme of the NO/cGMP signaling pathway. The heme moiety ligated to the beta-subunit via His(105) is crucial for the activation of the enzyme by NO. In addition to this NO binding capability, the heme status of the enzyme influences the activity of non-NO sGC activators and sGC inhibitors. Different sGC activity profiles were observed in the presence, absence, or the oxidized form of heme. Modulating the heme status is therefore crucial for the investigation of the mechanism of sGC activation. Here, we present a simple and reliable procedure for the removal of the heme moiety of sGC that is capable of eliminating any traces of unbound heme and detergent from the sample mixture in one single step. Samples containing 15 microg sGC and the non-ionic detergent Tween 20 (2%) were incubated at 37 degrees C for 10 min and loaded onto centrifugal ion exchange columns. After centrifugation, heme was bound entirely to the ion exchanger and could not be eluted, even after incubation with 1M NaCl. Tween 20 was found completely within the flowthrough. Heme-free sGC was eluted from the ion exchanger after application of 300 mM NaCl. The absence of the heme moiety was confirmed by UV/Vis spectra and determination of the enzymatic activity. In summary, the described procedure is suitable for the preparation of very small amounts of highly purified heme-free sGC for the investigation of the mechanism of action of different types of sGC activators.

Mechanisms of nitric oxide independent activation of soluble guanylyl cyclase.

The heterodimeric heme-protein soluble guanylyl cyclase (sGC) is the only proven receptor for nitric oxide (NO). Recently, two different types of NO-independent soluble guanylyl cyclase stimulators have been discovered. The heme-dependent stimulator 2-[1-[2-fluorophenyl)methyl]-1H-pyrazolo[3,4-b]pyridin-3-yl]-5(4-morpholinyl)-4,6-pyrimidinediamine (BAY 41-8543) stimulates the enzyme in a synergistic fashion when combined with NO, requires the presence of the heme group and can be blocked by the soluble guanylyl cyclase inhibitor 1H-(1,2,4)-Oxadiazole-(4,3-a)-quinoxalin-1-one (ODQ). The heme-independent activator 4-[((4-carboxybutyl)[2-[(4-phenethylbenzol) oxy]phenethyl]amino)methyl[benzoic]acid (BAY 58-2667) activates soluble guanylyl cyclase even in the presence of ODQ or rendered heme-deficient. In the present study, BAY 41-8543, BAY 58-2667 and NO strongly increased V(max). Combination of BAY 58-2667 and NO increased V(max) in an additive manner, whereas the synergistic effect of BAY 41-8543 and NO on enzyme activation was reflected in an overadditive increase of V(max). ODQ potentiated V(max) of BAY 58-2667-stimulated soluble guanylyl cyclase. BAY 41-8543 prolonged the half-life of the nitrosyl-heme complex of NO-activated enzyme, an effect that was not observed with BAY 58-2667. These results show the different activation patterns of both compounds and demonstrate their value as tools to investigate the mechanisms that underlie soluble guanylyl cyclase activation.

Overexpression of PH-4, a novel putative proline 4-hydroxylase, modulates activity of hypoxia-inducible transcription factors.

Hypoxia-inducible transcription factors (HIFs) are important for transcriptional adaptation to hypoxia. Availability of HIFs is regulated via posttranslational modification of their alpha subunits (HIF-1alpha and HIF-2alpha). Under normoxia, two highly conserved proline residues within the oxygen-dependent degradation domain (ODDD) are hydroxylated by oxoglutarate-dependent proline 4-hydroxylases EGLN1-3. Hydroxylated HIF-alpha interacts with the pVHL-E3 ubiquitin ligase complex and, subsequently, is degraded via the proteasomal pathway. We identified a novel putative proline 4-hydroxylase, PH-4, with an aminoterminal EF-hand motif and a carboxyterminal catalytic domain, which was highly expressed in most organs, and-unlike EGLNs which localize to the cytoplasm and nucleus-was associated with the endoplasmic reticulum. Like EGLNs, PH-4 overexpressed in cellular reporter assays suppressed the HIF transactivation activity, dependent on the consensus ODDD proline residues. Suppression of transactivation was correlated with decrease of cellular contents of HIF. Thus, PH-4 might be related to cellular oxygen sensing.

NO- and haem-independent activation of soluble guanylyl cyclase: molecular basis and cardiovascular implications of a new pharmacological principle.

1. Soluble guanylyl cyclase (sGC) is the only proven receptor for the ubiquitous biological messenger nitric oxide (NO) and is intimately involved in many signal transduction pathways, most notably in regulating vascular tone and platelet function. sGC is a heterodimeric (alpha/ss) protein that converts GTP to cyclic GMP; NO binds to its prosthetic haem group. Here, we report the discovery of a novel sGC activating compound, its interaction with a previously unrecognized regulatory site and its therapeutic implications. 2. Through a high-throughput screen we identified BAY 58-2667, an amino dicarboxylic acid which potently activates sGC in an NO-independent manner. In contrast to NO, YC-1 and BAY 41-2272, the sGC stimulators described recently, BAY 58-2667 activates the enzyme even after it has been oxidized by the sGC inhibitor ODQ or rendered haem deficient. 3. Binding studies with radiolabelled BAY 58-2667 show a high affinity site on the enzyme. 4. Using photoaffinity labelling studies we identified the amino acids 371 (alpha-subunit) and 231 - 310 (ss-subunit) as target regions for BAY 58-2667. 5. sGC activation by BAY 58-2667 results in an antiplatelet activity both in vitro and in vivo and a potent vasorelaxation which is not influenced by nitrate tolerance. 6. BAY 58-2667 shows a potent antihypertensive effect in conscious spontaneously hypertensive rats. In anaesthetized dogs the hemodynamic effects of BAY 58-2667 and GTN are very similar on the arterial and venous system. 7. This novel type of sGC activator is a valuable research tool and may offer a new approach for treating cardiovascular diseases.

Efomycine M, a new specific inhibitor of selectin, impairs leukocyte adhesion and alleviates cutaneous inflammation.

Specific interference with molecular mechanisms guiding tissue localization of leukocytes may be of great utility for selective immunosuppressive therapies. We have discovered and characterized efomycines, a new family of selective small-molecule inhibitors of selectin functions. Members of this family significantly inhibited leukocyte adhesion in vitro. Efomycine M, which was nontoxic and showed the most selective inhibitory effects on selectin-mediated leukocyte-endothelial adhesion in vitro, significantly diminished rolling in mouse ear venules in vivo as seen by intravital microscopy. In addition, efomycine M alleviated cutaneous inflammation in two complementary mouse models of psoriasis, one of the most common chronic inflammatory skin disorders. Molecular modeling demonstrated a spatial conformation of efomycines mimicking naturally occurring selectin ligands. Efomycine M might be efficacious in the treatment of human inflammatory disorders through a similar mechanism.

Pharmacological actions of a novel NO-independent guanylyl cyclase stimulator, BAY 41-8543: in vitro studies.

BAY 41-8543 is a novel, highly specific and so far the most potent NO-independent stimulator of sGC. Here we report the effects of BAY 41-8543 on the isolated enzyme, endothelial cells, platelets, isolated vessels and Langendorff heart preparation. BAY 41-8543 stimulates the recombinant sGC concentration-dependently from 0.0001 microM to 100 microM up to 92-fold. In combination, BAY 41-8543 and NO have synergistic effects over a wide range of concentrations. Similar results are shown in implying that BAY 41-8543 stimulates the sGC directly and furthermore makes the enzyme more sensitive to its endogenous activator NO. In vitro, BAY 41-8543 is a potent relaxing agent of aortas, saphenous arteries, coronary arteries and veins with IC(50)-values in the nM range. In the rat heart Langendorff preparation, BAY 41-8543 potently reduces coronary perfusion pressure from 10(-9) to 10(-6) g ml(-1) without any effect on left ventricular pressure and heart rate. BAY 41-8543 is effective even under nitrate tolerance conditions proved by the same vasorelaxing effect on aortic rings taken either from normal or nitrate-tolerant rats. BAY 41-8543 is a potent inhibitor of collagen-mediated aggregation in washed human platelets (IC(50)=0.09 microM). In plasma, BAY 41-8543 inhibits collagen-mediated aggregation better than ADP-induced aggregation, but has no effect on the thrombin pathway. BAY 41-8543 is also a potent direct stimulator of the cyclic GMP/PKG/VASP pathway in platelets and synergizes with NO over a wide range of concentrations. These results suggest that BAY 41-8543 is on the one hand an invaluable tool for studying sGC signaling in vitro and on the other hand its unique profile may offer a novel approach for treating cardiovascular diseases.

Cardiovascular actions of a novel NO-independent guanylyl cyclase stimulator, BAY 41-8543: in vivo studies.

BAY 41-8543 is a novel non-NO-based stimulator of sGC. This study investigates the acute effects of BAY 41-8543 on haemodynamics in anaesthetized rats and dogs, its long-term effects in conscious hypertension rat models and its antiplatelet effects. In anaesthetized dogs, intravenous injections of BAY 41-8543 (3 - 100 microg kg(-1)) caused a dose-dependent decrease in blood pressure and cardiac oxygen consumption as well as an increase in coronary blood flow and heart rate. In anaesthetized normotensive rats, BAY 41-8543 produced a dose-dependent and long-lasting blood pressure lowering effect after intravenous (3 - 300 microg kg(-1)) and oral (0.1 - 1 mg kg(-1)) administration. A dose-dependent and long-lasting decrease in blood pressure was also observed in conscious spontaneously hypertensive rats with a threshold dose of 0.1 mg kg(-1) p.o. After 3 mg kg(-1) the antihypertensive effect lasted for nearly 24 h. After multiple dosages, BAY 41-8543 did not develop tachyphylaxis in SHR. BAY 41-8543 prolonged the rat tail bleeding time and reduced thrombosis in the FeCl(3) thrombosis model after oral administration. In a low NO, high renin rat model of hypertension, BAY 41-8543 prevented the increase in blood pressure evoked by L-NAME and reveals a kidney protective effect. In this model, the overall beneficial effects of BAY 41-8543 manifested as both antiplatelet effect and vasodilatation were reflected in a significant reduction in mortality. The pharmacological profile of BAY 41-8543 suggests therefore that this compound has the potential to be an important research tool for in vivo investigations in the sGC/cGMP field and it also has the potential of being a unique clinical utility for treatment of cardiovascular diseases.