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Cytochrome P450-derived epoxyeicosatrienoic acids (EETs) stimulate endothelial cell proliferation and angiogenesis. In this study, we investigated the involvement of the forkhead box, class O (FOXO) family of transcription factors and their downstream target p27Kip1 in EET-induced endothelial cell proliferation. Incubation of human umbilical vein endothelial cells with 11,12-EET induced a time- and dose-dependent decrease in p27Kip1 protein expression, whereas p21Cip1 was not significantly affected. This effect on p27Kip1 protein was associated with decreased mRNA levels as well as p27Kip1 promoter activity. 11,12-EET also stimulated the time-dependent phosphorylation of Akt and of the forkhead factors FOXO1 and FOXO3a, effects prevented by the phosphatidylinositol 3-kinase inhibitor LY 294002. Transfection of endothelial cells with either a dominant-negative or an “Akt-resistant”/constitutively active FOXO3a mutant reversed the 11,12-EET-induced down-regulation of p27Kip1, whereas transfection of a constitutive active Akt decreased p27Kip1 expression independently of the presence or absence of 11,12-EET. To determine whether these effects are involved in EET-induced proliferation, endothelial cells were transfected with the 11,12-EET-generating epoxygenase CYP2C9. Transfection of CYP2C9 elicited endothelial cell proliferation and this effect was inhibited in cells co-transfected with CYP2C9 and either a dominant-negative Akt or constitutively active FOXO3a. Reducing FOXO expression using RNA interference, on the other hand, attenuated p27Kip1 expression and stimulated endothelial cell proliferation. These results indicate that EET-induced endothelial cell proliferation is associated with the phosphatidylinositol 3-kinase/Akt-dependent phosphorylation and inactivation of FOXO factors and the subsequent decrease in expression of the cyclin-dependent kinase inhibitor p27Kip1.
Die Häufigkeitsrate atopischer Erkrankungen bei Kindern, wie Heuschnupfen, Asthma, Neurodermitis (atopische Dermatitis), nimmt weltweit zu. Die Gründe sind vielschichtig. Gesichert ist der Zusammenhang zwischen der erblichen Überempfindlichkeit gegenüber natürlichen Substanzen (Atopie) und vermehrter Allergen- und Passivrauch-Exposition sowie Zunahme der Ein-Kind-Familien, Veränderung der mikrobiologischen Besiedlung des Darmes und Infektexposition. Besonders gut untersucht wurden diese Zusammenhänge von Erika von Mutius in einer Studie, in der sie von 1991 bis 1992 die Häufigkeit von Asthma in München (5030 Kinder) und Leipzig/Bitterfeld (2623 Kinder) verglichen hat.
Renal mesangial cells express high levels of matrix metalloproteinase 9 (MMP-9) in response to inflammatory cytokines such as interleukin (IL)-1β. We demonstrate here that the stable ATP analog adenosine 5′-O-(thiotriphosphate) (ATPγS) potently amplifies the cytokine-induced gelatinolytic content of mesangial cells mainly by an increase in the MMP-9 steady-state mRNA level. A Luciferase reporter gene containing 1.3 kb of the MMP-9 5′-promoter region showed weak responses to ATPγS but confered a strong ATP-dependent increase in Luciferase activity when under the additional control of the 3′-untranslated region of MMP-9. By in vitro degradation assay and actinomycin D experiments we found that ATPγS potently delayed the decay of MMP-9 mRNA. Gel-shift and supershift assays demonstrated that three AU-rich elements (AREs) present in the 3′-untranslated region of MMP-9 are constitutively bound by complexes containing the mRNA stabilizing factor HuR. The RNA binding of these complexes was markedly increased by ATPγS. Mutation of each ARE element strongly impaired the RNA binding of the HuR containing complexes. Reporter gene assays revealed that mutation of one ARE did not affect the stimulatory effects by ATPγS, but mutation of all three ARE motifs caused a loss of ATP-dependent increase in luciferase activity without affecting IL-1β-inducibility. By confocal microscopy we demonstrate that ATPγS increased the nucleo cytoplasmic shuttling of HuR and caused an increase in the cytosolic HuR level as shown by cell fractionation experiments. Together, our results indicate that the amplification of MMP-9 expression by extracellular ATP is triggered through mechanisms that likely involve a HuR-dependent rise in MMP-9 mRNA stability.
Biglycan, a nitric oxide-regulated gene, affects adhesion, growth, and survival of mesangial cells
(2003)
During glomerular inflammation mesangial cells are the major source and target of nitric oxide that pro-foundly influences proliferation, adhesion, and death of mesangial cells. The effect of nitric oxide on the mRNA expression pattern of cultured rat mesangial cells was therefore investigated by RNA-arbitrarily-primed polymerase chain reaction. Employing this approach, biglycan expression turned out to be down-regulated time- and dose-dependently either by interleukin-1beta-stimulated endogenous nitric oxide production or by direct application of the exogenous nitric oxide donor, diethylenetriamine nitric oxide. There was a corresponding decline in the rate of biglycan biosynthesis and in the steady state level of this proteoglycan. In vivo, in a model of mesangioproliferative glomerulonephritis up-regulation of inducible nitric-oxide synthase mRNA was associated with reduced expression of biglycan in isolated glomeruli. Biglycan expression could be normalized, both in vitro and in vivo, by using a specific inhibitor of the inducible nitric-oxide synthase, l-N6-(l-iminoethyl)-l-lysine dihydrochloride. Further studies showed that biglycan inhibited cell adhesion on type I collagen and fibronectin because of its binding to these substrates. More importantly, biglycan protected mesangial cells from apoptosis by decreasing caspase-3 activity, and it counteracted the proliferative effects of platelet-derived growth factor-BB. These findings indicate a signaling role of biglycan and describe a novel pathomechanism by which nitric oxide modulates the course of renal glomerular disease through regulation of biglycan expression.
Die Frankfurter Universitätsklinik hat mit der Eröffnung des interdisziplinären Brustkrebszentrums 1997 im Rhein-Main-Gebiet neue Maßstäbe bei der Behandlung von Brustkrebs gesetzt. Ziel ist es, die diagnostischen und therapeutischen Abläufe in der Brustkrebsbehandlung zu optimieren sowie die ökonomischen und fachlichen Ressourcen besser zu nutzen. Doch eine gute Therapie ist nur ein Werkzeug bei der Bekämpfung der seit Jahren zunehmenden Brustkrebserkrankungen. Nach Kaufmanns Ansicht ist es wichtig, "zweigleisig zu fahren: Früherkennungsmaßnahmen tragen dazu bei, Tumoren früh zu erkennen. Darüber hinaus gilt es, durch mehr Information mehr Körper- und Gesundheitsbewusstsein zu entwickeln. Denn wer gut informiert ist, hat die besseren Chancen."
The renin-angiotensin-aldosterone system plays a pivotal role in the regulation of salt and water homeostasis. Here, we demonstrate the expression and functional role of cGMP-dependent protein kinases (PKGs) in rat adrenal cortex. Expression of PKG II is restricted to adrenal zona glomerulosa (ZG) cells, whereas PKG I is localized to the adrenal capsule and blood vessels. Activation of the aldosterone system by a low sodium diet up-regulated the expression of PKG II, however, it did not change PKG I expression in adrenal cortex. Both, activation of PKG II in isolated ZG cell and adenoviral gene transfer of wild type PKG II into ZG cells enhanced aldosterone production. In contrast, inhibition of PKG II as well as infection with a PKG II catalytically inactive mutant had an inhibitory effect on aldosterone production. Steroidogenic acute regulatory (StAR) protein that regulates the rate-limiting step in steroidogenesis is a new substrate for PKG II and can be phosphorylated by PKG II in vitro at serine 55/56 and serine 99. Stimulation of aldosterone production by PKG II in contrast to stimulation by PKA did not activate StAR gene expression in ZG cells. The results presented indicate that PKG II activity in ZG cells is important for maintaining basal aldosterone production.
Cholecystokinin (CCK) and related peptides are potent growth factors in the gastrointestinal tract and may be important for human cancer. CCK exerts its growth modulatory effects through G(q)-coupled receptors (CCK(A) and CCK(B)) and activation of extracellular signal-regulated protein kinase 1/2 (ERK1/2). In the present study, we investigated the different mechanisms participating in CCK-induced activation of ERK1/2 in pancreatic AR42J cells expressing both CCK(A) and CCK(B). CCK activated ERK1/2 and Raf-1 to a similar extent as epidermal growth factor (EGF). Inhibition of EGF receptor (EGFR) tyrosine kinase or expression of dominant-negative Ras reduced CCK-induced ERK1/2 activation, indicating participation of the EGFR and Ras in CCK-induced ERK1/2 activation. However, compared with EGF, CCK caused only small increases in tyrosine phosphorylation of the EGFR and Shc, Shc-Grb2 complex formation, and Ras activation. Signal amplification between Ras and Raf in a CCK-induced ERK cascade appears to be mediated by activation of protein kinase Cepsilon (PKCepsilon), because 1) down-modulation of phorbol ester-sensitive PKCs inhibited CCK-induced activation of Ras, Raf, and ERK1/2 without influencing Shc-Grb2 complex formation; 2) PKCepsilon, but not PKCalpha or PKCdelta, was detectable in Raf-1 immunoprecipitates, although CCK activated all three PKC isoenzymes. In addition, the present study provides evidence that the Src family tyrosine kinase Yes is activated by CCK and mediates CCK-induced tyrosine phosphorylation of Shc. Furthermore, we show that CCK-induced activation of the EGFR and Yes is achieved through the CCK(B) receptor. Together, our data show that different signals emanating from the CCK receptors mediate ERK1/2 activation; activation of Yes and the EGFR mediate Shc-Grb2 recruitment, and activation of PKC, most likely PKCepsilon, augments CCK-stimulated ERK1/2 activation at the Ras/Raf level.
AIM: To evaluate and compare the effect of combined transarterial chemoembolization (TACE) and arterial administration of Bletilla striata (a Chinese traditional medicine against liver tumor) versus TACE alone for the treatment of hepatocellular carcinoma (HCC) in ACI rats.
METHODS: Subcapsular implantation of a solid Morris hepatoma 3 924A (2 mm3) in the liver was carried out in 30 male ACI rats. Tumor volume (V1) was measured by magnetic resonance imaging (MRI) on day 13 after implantation. The following different agents of interventional treatment were injected after retrograde catheterization via gastroduodenal artery (on day 14), namely, (A) TACE (0.1 mg mitomycin + 0.1 ml Lipiodol) + Bletilla striata (1.0 mg) (n=10); (B) TACE + Bletilla striata (1.0 mg) + ligation of hepatic artery (n=10), (C) TACE alone (control group, n=10). Tumor volume (V2) was assessed by MRI (on day 13 after treatment) and the tumor growth ratio (V2/V1) was calculated.
RESULTS: The mean tumor volume before (V1) and after (V2) treatment was 0.0355 cm3 and 0.2248 cm3 in group A, 0.0374 cm3 and 0.0573 cm3 in group B, 0.0380 cm3 and 0.3674 cm3 in group C, respectively. The mean ratio (V2/V1) was 6.2791 in group A, 1.5324 in group B and 9.1382 in group C. Compared with the control group (group C), group B showed significant inhibition of tumor growth (P<0.01), while group A did not (P>0.05). None of the animals died during implantation or in the postoperative period.
CONCLUSION: Combination of TACE and arterial administration of Bletilla striata plus ligation of hepatic artery is more effective than TACE alone in the treatment of HCC in rats.
Hinter dem Begriff "Schlaganfall" verbergen sich verschiedene Krankheitsbilder, die durch gemeinsame Merkmale gekennzeichnet sind: Die Beschwerden treten akut auf, oftmals von einer Sekunde zur anderen. Ein Schlagfall ist darüber hinaus durch das Auftreten von charakteristischen neurologischen Symptomen gekennzeichnet, wie halbseitige Lähmungen, Sprach-, Seh- oder Gefühlsstörungen. Die Ursache hierfür liegt in Veränderungen in den Blutgefäßen des Gehirns, wie die Autoren erläutern.
Glukose ist ein zentrales Element des normalen Energiehaushalts im Körper. Seine Konzentration im Blut wird von Insulin, einem Hormon, das außerdem für Wachstums- und Entwicklungsprozesse mitverantwortlich ist, reguliert. Normale Glukosespiegel im Blut und in den Zellen sind die Folge einer fein abgestimmten Insulinwirkung am Insulinrezeptor, der in der Zelle eine nachgeschaltete Signalkette auslöst, bei der Glukose in die Zelle eingeschleust wird. Insulin bindet an den Insulinrezeptor nach dem Schlüssel-Schloss-Prinzip, wobei die Türöffnung hier den Eintritt von Glukose in die Zelle bedeutet. Gibt es zu wenig Insulin (Typ 1-Diabetes) oder ist die Wirkung von Insulin am Rezeptor oder in der nachgeschalteten Signalwirkung gestört, so resultieren Zuckerstoffwechselveränderungen bis hin zu Diabetes mellitus (Zuckerkrankheit). Für die häufigste Diabetes mellitus-Form (Typ 2) sind Störungen am Rezeptorsystem verantwortlich.
Die Fettleibigkeit nimmt in Europa in alarmierender Weise zu und ist deshalb von der Weltgesundheitsorganisation (WHO) als eine weltweite Epidemie eingestuft worden. Für die europäische Vereinigung zur Untersuchung der Obesitas (European Association for the Study of Obesity, EASO), in der Grundlagenforscher, Kliniker und Epidemiologen zusammenarbeiten, gilt die Fettsucht als "wichtigste Barriere zur Prävention chronischer, nicht-übertragbarer Krankheiten". In vielen europäischen Ländern ist mehr als die Hälfte der Bevölkerung übergewichtig und bis zu 30 Prozent der Bevölkerung sind fettleibig. Die Prävalenz bei Kindern ist deutlich ansteigend, so dass in einigen Regionen nahezu jedes vierte Kind betroffen ist.
Im März 2000 hat der Europäische Rat von Lissabon das strategische Ziel der EU formuliert, "die Union zum wettbewerbsfähigsten und dynamischsten wissensbasierten Wirtschaftsraum der Welt zu machen – ... der fähig ist, ein dauerhaftes Wirtschaftswachstum mit mehr und besseren Arbeitsplätzen und einem größeren sozialen Zusammenhalt zu schaffen". Die Umsetzung soll u. a. durch die "Offene Methode der Koordinierung" (OMK) vorangetrieben werden. Damit wird eine Leitungs- und Koordinierungsfunktion des Rates auch auf solchen Politikfeldern angestrebt, die nach dem Vertragsrecht in die alleinige Gestaltungskompetenz der Mitgliedstaaten fallen. Die OMK lässt sich als eine Art Benchmarking-Verfahren ohne unmittelbar verpflichtende Konsequenzen für die Mitgliedstaaten beschreiben. Ein ähnliches Vorgehen wird seit 1997 in der Beschäftigungspolitik praktiziert. Mit der Lissabon-Strategie wird die OMK sukzessive auf weitere sozialpolitische Bereiche ausgedehnt. Im Bereich der Alterssicherung kommt sie ebenso zum Einsatz wie bei der Bekämpfung von Armut und sozialer Ausgrenzung. Künftig wird auch die Gesundheitspolitik der Mitgliedsländer auf dem Prüfstand der OMK stehen.
Endothelium-dependent vasodilation is thought to be mediated primarily by the NO/cGMP signaling pathway whereas cAMP-elevating vasodilators are considered to act independent of the endothelial cell layer. However, recent functional data suggest that cAMP-elevating vasodilators such as β-receptor agonists, adenosine or forskolin may also be endothelium-dependent. Here we used functional and biochemical assays to analyze endothelium-dependent, cGMP- and cAMP-mediated signaling in rat aorta. Acetylcholine and sodium nitroprusside (SNP) induced a concentration-dependent relaxation of phenylephrine-precontracted aorta. This response was reflected by the phosphorylation of the vasodilator-stimulated phosphoprotein (VASP), a validated substrate of cGMP- and cAMP-dependent protein kinases (cGK, cAK), on Ser157 and Ser239. As expected, the effects of acetylcholine were endothelium-dependent. However, relaxation induced by the β-receptor agonist isoproterenol was also almost completely impaired after endothelial denudation. At the biochemical level, acetylcholine- and isoproterenol-evoked cGK and cAK activation, respectively, as measured by VASP Ser239 and Ser157 phosphorylation, was strongly diminished. Furthermore, the effects of isoproterenol were repressed by eNOS inhibition when endothelium was present. We also observed that the relaxing and biochemical effects of forskolin were at least partially endothelium-dependent. We conclude that cAMP-elevating vasodilators, i.e. isoproterenol and to a lesser extent also forskolin, induce vasodilation and concomitant cyclic nucleotide protein kinase activation in the vessel wall in an endothelium-dependent way.
Atrial natriuretic peptide (ANP) plays a key regulatory role in arterial blood pressure homeostasis. We recently generated mice with selective deletion of the ANP receptor, guanylyl cyclase-A (GC-A), in vascular smooth muscle (SMC GC-A knockout (KO) mice) and reported that resting arterial blood pressure was completely normal in spite of clear abolition of the direct vasodilating effects of ANP (Holtwick, R., Gotthardt, M., Skryabin, B., Steinmetz, M., Potthast, R., Zetsche, B., Hammer, R. E., Herz, J., and Kuhn M. (2002) Proc. Natl. Acad. Sci. U. S. A. 99, 7142–7147). The purpose of this study was to clarify mechanisms compensating for the missing vasodilator responses to ANP. In particular, we analyzed the effect of the endothelial, cGMP-mediated vasodilators C-type natriuretic peptide and nitric oxide (NO). In isolated arteries from SMC GC-A KO mice, the vasorelaxing sensitivity to sodium nitroprusside and the endothelium-dependent vasodilator, acetylcholine, was significantly greater than in control mice. There was no difference in responses to C-type natriuretic peptide or to the activator of cGMP-dependent protein kinase I, 8-para-chlorophenylthio-cGMP. The aortic expression of soluble GC (sGC), but not of endothelial NO synthase or cGMP-dependent protein kinase I, was significantly increased in SMC GC-A KO mice. Chronic oral treatment with the NO synthase inhibitor Nw-nitro-l-arginine methyl ester increased arterial blood pressure, the effect being significantly enhanced in SMC GC-A KO mice. We conclude that SMC GC-A KO mice exhibit a higher vasodilating sensitivity to NO. This can be attributed to an enhanced expression of sGC, whereas the expression and/or activity levels of downstream cGMP-effector pathways are not involved. Increased vasodilating responsiveness to endothelial NO contributes to compensate for the missing vasodilating effect of ANP in SMC GC-A KO mice.
Klassenmedizin
(2003)
Atovaquone is a substituted 2-hydroxynaphthoquinone that is used therapeutically to treat Plasmodium falciparum malaria, Pneumocystis carinii pneumonia, and Toxoplasma gondii toxoplasmosis. It is thought to act on these organisms by inhibiting the cytochrome bc1 complex. We have examined the interaction of atovaquone with the bc1 complex isolated from Saccharomyces cerevisiae, a surrogate, nonpathogenic fungus. Atovaquone inhibits the bc1 complex competitively with apparent Ki = 9 nm, raises the midpoint potential of the Rieske iron-sulfur protein from 285 to 385 mV, and shifts the g values in the EPR spectrum of the Rieske center. These results indicate that atovaquone binds to the ubiquinol oxidation pocket of the bc1 complex, where it interacts with the Rieske iron-sulfur protein. A computed energy-minimized structure for atovaquone liganded to the yeast bc1 complex suggests that a phenylalanine at position 275 of cytochrome b in the bovine bc1 complex, as opposed to leucine at the equivalent position in the yeast enzyme, is responsible for the decreased sensitivity of the bovine bc1 complex (Ki = 80 nm) to atovaquone. When a L275F mutation was introduced into the yeast cytochrome b, the sensitivity of the yeast enzyme to atovaquone decreased (Ki = 100 nm) with no loss in activity, confirming that the L275F exchange contributes to the differential sensitivity of these two species to atovaquone. These results provide the first molecular description of how atovaquone binds to the bc1 complex and explain the differential inhibition of the fungal versus mammalian enzymes.
Signal transducer and activator of transcription 5 (STAT5) is a transcription factor that activates prolactin (PRL)-dependent gene expression in the mammary gland. For the activation of its target genes, STAT5 recruits coactivators like p300 and the CREB-binding protein (CBP). In this study we analyzed the function of p300/CBP-associated members of the p160/SRC/NCoA-family in STAT5-mediated transactivation of β-casein expression. We found that only one of them, NCoA-1, acts as a coactivator for both STAT5a and STAT5b. The two coactivators p300/CBP and NCoA-1 cooperatively enhance STAT5a-mediated transactivation. For NCoA-1-dependent coactivation of STAT5, both the activation domain 1 and the amino-terminal bHLH/PAS domain are required. The amino-terminal region mediates the interaction with STAT5a in cells. A motif of three amino acids in an α-helical region of the STAT5a-transactivation domain is essential for the binding of NCoA-1 and for the transcriptional activity of STAT5a. Moreover we observed that NCoA-1 is involved in the synergistic action of the glucocorticoid receptor and STAT5a on the β-casein promoter. These findings support a model in which STAT5, in concert with the glucocorticoid receptor, recruits a multifunctional coactivator complex to initiate the PRL-dependent transcription.
Mismatch repair is a highly conserved system that ensures replication fidelity by repairing mispairs after DNA synthesis. In humans, the two protein heterodimers hMutSα (hMSH2‐hMSH6) and hMutLα (hMLH1‐hPMS2) constitute the centre of the repair reaction. After recognising a DNA replication error, hMutSα recruits hMutLα, which then is thought to transduce the repair signal to the excision machinery. We have expressed an ATPase mutant of hMutLα as well as its individual subunits hMLH1 and hPMS2 and fragments of hMLH1, followed by examination of their interaction properties with hMutSα using a novel interaction assay. We show that, although the interaction requires ATP, hMutLα does not need to hydrolyse this nucleotide to join hMutSα on DNA, suggesting that ATP hydrolysis by hMutLα happens downstream of complex formation. The analysis of the individual subunits of hMutLα demonstrated that the hMutSα–hMutLα interaction is predominantly conferred by hMLH1. Further experiments revealed that only the N‐terminus of hMLH1 confers this interaction. In contrast, only the C‐terminus stabilised and co‐immunoprecipitated hPMS2 when both proteins were co‐expressed in 293T cells, indicating that dimerisation and stabilisation are mediated by the C‐terminal part of hMLH1. We also examined another human homologue of bacterial MutL, hMutLβ (hMLH1–hPMS1). We show that hMutLβ interacts as efficiently with hMutSα as hMutLα, and that it predominantly binds to hMutSα via hMLH1 as well.
Introduction: This open label, multicentre study was conducted to assess the times to offset of the pharmacodynamic effects and the safety of remifentanil in patients with varying degrees of renal impairment requiring intensive care.
Methods: A total of 40 patients, who were aged 18 years or older and had normal/mildly impaired renal function (estimated creatinine clearance ≥ 50 ml/min; n = 10) or moderate/severe renal impairment (estimated creatinine clearance <50 ml/min; n = 30), were entered into the study. Remifentanil was infused for up to 72 hours (initial rate 6–9 μg/kg per hour), with propofol administered if required, to achieve a target Sedation–Agitation Scale score of 2–4, with no or mild pain.
Results: There was no evidence of increased offset time with increased duration of exposure to remifentanil in either group. The time to offset of the effects of remifentanil (at 8, 24, 48 and 72 hours during scheduled down-titrations of the infusion) were more variable and were statistically significantly longer in the moderate/severe group than in the normal/mild group at 24 hours and 72 hours. These observed differences were not clinically significant (the difference in mean offset at 72 hours was only 16.5 min). Propofol consumption was lower with the remifentanil based technique than with hypnotic based sedative techniques. There were no statistically significant differences between the renal function groups in the incidence of adverse events, and no deaths were attributable to remifentanil use.
Conclusion: Remifentanil was well tolerated, and the offset of pharmacodynamic effects was not prolonged either as a result of renal dysfunction or prolonged infusion up to 72 hours.
There is evidence that endothelial nitric-oxide synthase (eNOS) is regulated by reciprocal dephosphorylation of Thr497 and phosphorylation of Ser1179. To examine the interrelationship between these sites, cells were transfected with wild-type (WT), T497A, T497D, S1179D, and T497A/S1179D eNOS and activity, NO release and eNOS localization were assessed. Although eNOS T497A, S1179D and T497A/S1179D eNOS had greater enzymatic activity than did WT eNOS in lysates, basal production of NO from cells was markedly reduced in cells transfected with T497A and T497A/S1179D eNOS but augmented in cells transfected with S1179D eNOS. Stimulating cells with ATP or ionophore normalized the loss of function seen with T497A and T497A/S1179D eNOS to levels observed with WT and S1179D eNOS, respectively. Despite these functional differences, the localization of eNOS mutants were similar to WT. Because both T497A and T497A/S1179D eNOS exhibited higher enzyme activity but reduced production of NO, we examined whether these mutations were “uncoupling” NO synthesis. T497A and T497A/S1179D eNOS generated 2-3 times more superoxide anion than WT eNOS, and both basal and stimulated interactions of T497A/S1179D eNOS with hsp90 were reduced in co-immunoprecipitation experiments. Thus, the phosphorylation/dephosphorylation of Thr497 may be an intrinsic switch mechanism that determines whether eNOS generates NO versus superoxide in cells.
Neueren Schätzungen zufolge leiden bis zu 15 Prozent der bundesdeutschen Bevölkerung unter klinisch bedeutsamen Schlafstörungen. Die "Internationale Klassifikation der Schlafstörungen" (ICSD-R) umfasst insgesamt 88 Störungen, die sich vier Oberkategorien zuordnen lassen: "Dyssomnien" (Schlafstörungen, die entweder durch Ein- oder Durchschlafstörungen oder übermäßige Schläfrigkeit gekennzeichnet sind), "Parasomnien" (zum Beispiel Schlafwandeln oder Sprechen im Schlaf), "Schlafstörungen bei körperlichen oder psychiatrischen Erkrankungen" sowie "Vorgeschlagene Schlafstörungen" (diagnostische Kategorien, die derzeit noch näher erforscht werden).
Receptor tyrosine kinases of the epidermal growth factor (EGF) receptor family regulate essential cellular functions such as proliferation, survival, migration, and differentiation but also play central roles in the etiology and progression of tumors. We have identified short peptide sequences from a random peptide library integrated into the thioredoxin scaffold protein, which specifically bind to the intracellular domain of the EGF receptor (EGFR). These molecules have the potential to selectively inhibit specific aspects of EGF receptor signaling and might become valuable as anticancer agents. Intracellular expression of the aptamer encoding gene construct KDI1 or introduction of bacterially expressed KDI1 via a protein transduction domain into EGFR-expressing cells results in KDI1·EGF receptor complex formation, a slower proliferation, and reduced soft agar colony formation. Aptamer KDI1 did not summarily block the EGF receptor tyrosine kinase activity but selectively interfered with the EGF-induced phosphorylation of the tyrosine residues 845, 1068, and 1148 as well as the phosphorylation of tyrosine 317 of p46 Shc. EGF-induced phosphorylation of Stat3 at tyrosine 705 and Stat3-dependent transactivation were also impaired. Transduction of a short synthetic peptide aptamer sequence not embedded into the scaffold protein resulted in the same impairment of EGF-induced Stat3 activation.
Heute erkranken in Deutschland etwa 750 Kinder und Jugendliche Jahr für Jahr an Leukämien und anderen bösartigen Bluterkrankungen, davon rund 600 an einer Akuten Lymphatischen Leukämie (ALL) und 150 an einer Akuten Myeloischen Leukämie (AML). Seit der Einführung moderner Chemotherapieprotokolle im Jahr 1970 ist aus dieser einst tödlichen eine jetzt heilbare Erkrankung geworden. Allerdings erleiden immer noch zirka 20 Prozent der Kinder mit einer ALL einen Rückfall. Die meisten der Kinder benötigen eine Stammzelltransplantation. In Deutschland sind dies pro Jahr etwa 100; in Frankfurt werden im Jahr gegenwärtig 20 Patienten mit Stammzelltransplantationen behandelt, davon rund 55 Prozent mit akuten Leukämien. Eine Stammzell-transplantation kostet bei Kindern gegenwärtig etwa 120000 Euro.
Tetrahydrobiopterin, a critical factor in the production and role of nitric oxide in mast cells
(2003)
Mast cells (MC) are biologically potent, ubiquitously distributed immune cells with fundamental roles in host integrity and disease. MC diversity and function is regulated by exogenous nitric oxide; however, the production and function of endogenously produced NO in MC is enigmatic. We used rat peritoneal MC (PMC) as an in vivo model to examine intracellular NO production. Live cell confocal analysis of PMC using the NO-sensitive probe diaminofluorescein showed distinct patterns of intracellular NO formation with either antigen (Ag)/IgE (short term) or interferon-γ (IFN-γ) (long term). Ag/IgE-induced NO production is preceded by increased intracellular Ca2+, implying constitutive nitric-oxide synthase (NOS) activity. NO formation inhibits MC degranulation. NOS has obligate requirements for tetrahydrobiopterin (BH4), a product of GTP-cyclohydrolase I (CHI), IFN-γ-stimulated PMC increased CHI mRNA, protein, and enzymatic activity, while decreasing CHI feedback regulatory protein mRNA, causing sustained NO production. Treatment with the CHI inhibitor, 2,4-diamino-6-hydroxypyrimidine, inhibited NO in both IFN-γ and Ag/IgE systems, increasing MC degranulation. Reconstitution with the exogenous BH4 substrate, sepiapterin, restored NO formation and inhibited exocytosis. Thus, Ag/IgE and IFN-γ induced intracellular NO plays a key role in MC mediator release, and alterations in NOS activity via BH4 availability may be critical to the heterogeneous responsiveness of MC.
Mitochondrial proton-translocating NADH:ubiquinone oxidoreductase (complex I) couples the transfer of two electrons from NADH to ubiquinone to the translocation of four protons across the mitochondrial inner membrane. Subunit PSST is the most likely carrier of iron-sulfur cluster N2, which has been proposed to play a crucial role in ubiquinone reduction and proton pumping. To explore the function of this subunit we have generated site-directed mutants of all eight highly conserved acidic residues in the Yarrowia lipolytica homologue, the NUKM protein. Mutants D99N and D115N had only 5 and 8% of the wild type catalytic activity, respectively. In both cases complex I was stably assembled but electron paramagnetic resonance spectra of the purified enzyme showed a reduced N2 signal (about 50%). In terms of complex I catalytic activity, almost identical results were obtained when the aspartates were individually changed to glutamates or to glycines. Mutations of other conserved acidic residues had less dramatic effects on catalytic activity and did not prevent assembly of iron-sulfur cluster N2. This excludes all conserved acidic residues in the PSST subunit as fourth ligands of this redox center. The results are discussed in the light of the structural similarities to the homologous small subunit of water-soluble [NiFe] hydrogenases.