Synthese von antiinflammatorischen Makrolactonen des Oxacyclododecindion-Typs und deren biologische Evaluierung

Die Sekundärmetabolite 4-Dechlor-14-deoxyoxacyclododecindion, 14-Deoxyoxacyclo-dodecindion und Oxacyclododecindion zeigten in ersten in vitro-Studien eine Hemmung des TGF-β- sowie des JAK-STAT-Signaltransduktionsweges im nanomolaren Konzentrationsbereich. Sie stellen potentielle Leitstrukturen für die Entwicklung neuer Therapeutika zur Behandlung chronisch entzündlicher und/oder fibrotischer Erkrankungen dar. Ziel dieser Arbeit war die Entwicklung eines totalsynthetischen Zugangs zu diesen Makrolactonen.rnDer erste retrosynthetische Ansatz bestand aus einer Ringschluss-Metathese/Reduktions/Eliminierungs-Sequenz. Während das gesättigte Makrolacton-grundgerüst dargestellt werden konnte, schlug die Einführung der Doppelbindung fehl. Es wurde nur ein exo-Methylen-Derivat erhalten. Eine Syntheseroute über eine carbonylierende Kreuzkupplung oder über eine intramolekulare Hydroacylierung verliefen erfolglos. Versuche zum Aufbau des α,β-ungesättigten Enons über das β,γ-ungesättigte Enon in einer Ringschluss-Metathese/Isomerisierungs-Sequenz führten stattdessen zur Bildung eines γ,δ-ungesättigten Ketons und eines 12-Oxo-10,11-dehydrocurvularin-Derivates.rnEine intramolekulare Friedel-Crafts-Acylierung ermöglichte den Ringschluss, sodass die beiden Naturstoffe 4-Dechlor-14-deoxyoxacyclododecindion sowie 14-Deoxyoxa-cyclododecindion synthetisiert werden konnten. Durch die Totalsynthese konnte zudem die bisher unbekannte relative Konfiguration der zwei Stereozentren aufgeklärt werden. Die während dieser Arbeit erhaltenen Derivate ermöglichten es, Struktur-Wirkungs-Beziehungen für diese Makrolactone aufzustellen.rnIn weiteren biologischen Studien von Kooperationspartnern wurde die hohe Wirksamkeit im nanomolaren Konzentrationsbereich bestätigt. Eine erste in vivo-Studie zur Behandlung von systemischem Lupus erythematodes mit 14-Deoxyoxacyclododecindion deutet auf eine verminderte Entzündungsreaktion und positive Effekte auf chronische Nierenschäden hin.rn

The FGFRL1 receptor is shed from cell membranes, binds fibroblast growth factors (FGFs), and antagonizes FGF signaling in Xenopus embryos

FGFRL1 (fibroblast growth factor receptor like 1) is the fifth and most recently discovered member of the fibroblast growth factor receptor (FGFR) family. With up to 50% amino acid similarity, its extracellular domain closely resembles that of the four conventional FGFRs. Its intracellular domain, however, lacks the split tyrosine kinase domain needed for FGF-mediated signal transduction. During embryogenesis of the mouse, FGFRL1 is essential for the development of parts of the skeleton, the diaphragm muscle, the heart, and the metanephric kidney. Since its discovery, it has been hypothesized that FGFRL1 might act as a decoy receptor for FGF ligands. Here we present several lines of evidence that support this notion. We demonstrate that the FGFRL1 ectodomain is shed from the cell membrane of differentiating C2C12 myoblasts and from HEK293 cells by an as yet unidentified protease, which cuts the receptor in the membrane-proximal region. As determined by ligand dot blot analysis, cell-based binding assays, and surface plasmon resonance analysis, the soluble FGFRL1 ectodomain as well as the membrane-bound receptor are capable of binding to some FGF ligands with high affinity, including FGF2, FGF3, FGF4, FGF8, FGF10, and FGF22. We furthermore show that ectopic expression of FGFRL1 in Xenopus embryos antagonizes FGFR signaling during early development. Taken together, our data provide strong evidence that FGFRL1 is indeed a decoy receptor for FGFs.

Acute Regulation of Renal Na+/H+ Exchanger NHE3 by Dopamine: Role of Protein Phosphatase 2A

Nephrogenic dopamine is a potent natriuretic paracrine/autocrine hormone that is central for mammalian sodium homeostasis. In the renal proximal tubule, dopamine induces natriuresis partly via inhibition of the sodium/proton exchanger NHE3. The signal transduction pathways and mechanisms by which dopamine inhibits NHE3 are complex and incompletely understood. This manuscript describes the role of the serine/threonine protein phosphatase 2A (PP2A) in the regulation of NHE3 by dopamine. The PP2A regulatory subunit B56 delta (coded by the Ppp2r5d gene) directly associates with more than one region of the carboxy-terminal hydrophilic putative cytoplasmic domain of NHE3 (NHE3-cyto), as demonstrated by yeast-two-hybrid, co-immunoprecipitation, blot overlay and in vitro pull-down assays. Phosphorylated NHE3-cyto is a substrate for purified PP2A in an in vitro dephosphorylation reaction. In cultured renal cells, inhibition of PP2A by either okadaic acid or by overexpression of the simian virus 40 (SV40) small t antigen blocks the ability of dopamine to inhibit NHE3 activity and to reduce surface NHE3 protein. Dopamine-induced NHE3 redistribution is also blocked by okadaic acid ex vivo in rat kidney cortical slices. These studies demonstrate that PP2A is an integral and critical participant in the signal transduction pathway between dopamine receptor activation and NHE3 inhibition. Key words: Natriuresis, Sodium transport, Signal transduction.

Ephrin-B2 controls VEGF-induced angiogenesis and lymphangiogenesis

In development, tissue regeneration or certain diseases, angiogenic growth leads to the expansion of blood vessels and the lymphatic vasculature. This involves endothelial cell proliferation as well as angiogenic sprouting, in which a subset of cells, termed tip cells, acquires motile, invasive behaviour and extends filopodial protrusions. Although it is already appreciated that angiogenesis is triggered by tissue-derived signals, such as vascular endothelial growth factor (VEGF) family growth factors, the resulting signalling processes in endothelial cells are only partly understood. Here we show with genetic experiments in mouse and zebrafish that ephrin-B2, a transmembrane ligand for Eph receptor tyrosine kinases, promotes sprouting behaviour and motility in the angiogenic endothelium. We link this pro-angiogenic function to a crucial role of ephrin-B2 in the VEGF signalling pathway, which we have studied in detail for VEGFR3, the receptor for VEGF-C. In the absence of ephrin-B2, the internalization of VEGFR3 in cultured cells and mutant mice is defective, which compromises downstream signal transduction by the small GTPase Rac1, Akt and the mitogen-activated protein kinase Erk. Our results show that full VEGFR3 signalling is coupled to receptor internalization. Ephrin-B2 is a key regulator of this process and thereby controls angiogenic and lymphangiogenic growth.

Pineal melatonin synthesis is altered in Period1 deficient mice

Melatonin is an important endocrine signal for darkness in mammals. Transcriptional activation of the arylalkylamine-N-acetyltransferase gene encoding for the penultimate enzyme in melatonin synthesis drives the daily rhythm of the hormone in the pineal gland of rodents. Rhythmic arylalkylamine-N-acetyltransferase expression is controlled by the cAMP-signal transduction pathway and involves the activation of ?-adrenergic receptors and the inducible cAMP early repressor. In addition, the rat arylalkylamine-N-acetyltransferase promoter contains an E-box element which can interact with clock proteins. Moreover, the pineal gland of mice shows a circadian rhythm in clock proteins such as the transcriptional repressor Period1, which has been shown to control rhythmic gene expression in a variety of tissues. However, the role of Period1 in the regulation of pineal melatonin synthesis is still unknown. Therefore, circadian rhythms in arylalkylamine-N-acetyltransferase, ?-adrenergic receptor, and inducible cAMP early repressor mRNA levels (real time PCR), arylalkylamine-N-acetyltransferase enzyme activity (radiometric assay) and melatonin concentration radio immuno assay (RIA) were analyzed in the pineal gland of mice with a targeted deletion of the Period1 gene (Per1-/-) and the corresponding wildtype. In Per1-/- the amplitude in arylalkylamine-N-acetyltransferase expression was significantly elevated as compared to wildtype. In contrast, ?-adrenergic receptor and inducible cAMP early repressor mRNA levels were not affected by the Period1-deficiency. This indicates that the molecular clockwork alters the amplitude of arylalkylamine-N-acetyltransferase expression. In vitro, pineal glands of Per1-/- mice showed a day night difference in arylalkylamine-N-acetyltransferase expression with high levels at night. This suggests that a deficient in Period1 elicits similar effects as the activation of the cAMP-signal transduction pathway in wildtype mice.

Identification of candidate genes and physiological pathways involved in gonad deformation in whitefish (Coregonus spp.) from Lake Thun, Switzerland

In 2000, fishermen reported the appearance of deformed reproductive organs in whitefish (Coregonus spp.) from Lake Thun, Switzerland. Despite intensive investigations, the causes of these abnormalities remain unknown. Using gene expression profiling, we sought to identify candidate genes and physiological processes possibly associated with the observed gonadal deformations, in order to gain insights into potential causes. Using in situ-synthesized oligonucleotide arrays, we compared the expression levels at 21,492 unique transcript probes in liver and head kidney tissue of male whitefish with deformed and normally developed gonads, respectively. The fish had been collected on spawning sites of two genetically distinct whitefish forms of Lake Thun. We contrasted the gene expression profiles of 56 individuals, i.e., 14 individuals of each phenotype and of each population. Gene-by-gene analysis revealed weak expression differences between normal and deformed fish, and only one gene, ictacalcin, was found to be up-regulated in head kidney tissue of deformed fish from both whitefish forms, However, this difference could not be confirmed with quantitative real-time qPCR. Enrichment analysis on the level of physiological processes revealed (i) the involvement of immune response genes in both tissues, particularly those linked to complement activation in the liver, (ii) proteolysis in the liver and (iii) GTPase activity and Ras protein signal transduction in the head kidney. In comparison with current literature, this gene expression pattern signals a chronic autoimmune disease in the testes. Based on the recent observations that gonad deformations are induced through feeding of zooplankton from Lake Thun we hypothesize that a xenobiotic accumulated in whitefish via the plankton triggering autoimmunity as the likely cause of gonad deformations. We propose several experimental strategies to verify or reject this hypothesis.

Phosphatidylinositol 3-kinase isoforms as novel drug targets

Phosphatidylinositol 3-kinases (PI3Ks) are key molecules in the signal transduction pathways initiated by the binding of extracellular signals to their cell surface receptors. The PI3K family of enzymes comprises eight catalytic isoforms subdivided into three classes and control a variety of cellular processes including proliferation, growth, apoptosis, migration and metabolism. Deregulation of the PI3K pathway has been extensively investigated in connection to cancer, but is also involved in other commonly occurring diseases such as chronic inflammation, autoimmunity, allergy, atherosclerosis, cardiovascular and metabolic diseases. The fact that the PI3K pathway is deregulated in a large number of human diseases, and its importance for different cellular responses, makes it an attractive drug target. Pharmacological PI3K inhibitors have played a very important role in studying cellular responses involving these enzymes. Currently, a wide range of selective PI3K inhibitors have been tested in preclinical studies and some have entered clinical trials in oncology. However, due to the complexity of PI3K signaling pathways, developing an effective anti-cancer therapy may be difficult. The biggest challenge in curing cancer patients with various signaling pathway abnormalities is to target multiple components of different signal transduction pathways with mechanism-based combinatorial treatments. In this article we will give an overview of the complex role of PI3K isoforms in human diseases and discuss their potential as drug targets. In addition, we will describe the drugs currently used in clinical trials, as well as promising emerging candidates.

Eosinophil extracellular DNA traps: molecular mechanisms and potential roles in disease

Eosinophil extracellular traps (EETs) are part of the innate immune response and are seen in multiple infectious, allergic, and autoimmune eosinophilic diseases. EETs are composed of a meshwork of DNA fibers and eosinophil granule proteins, such as major basic protein (MBP) and eosinophil cationic protein (ECP). Interestingly, the DNA within the EETs appears to have its origin in the mitochondria of eosinophils, which had released most their mitochondrial DNA, but were still viable, exhibiting no evidence of a reduced life span. Multiple eosinophil activation mechanisms are represented, whereby toll-like, cytokine, chemokine, and adhesion receptors can all initiate transmembrane signal transduction processes leading to the formation of EETs. One of the key signaling events required for DNA release is the activation of the NADPH oxidase. Here, we review recent progress made in the understanding the molecular mechanisms involved in DNA and granule protein release, discuss the presence of EETs in disease, speculate on their potential role(s) in pathogenesis, and compare available data on other DNA-releasing cells, particularly neutrophils.

Atomic force microscopy for the study of membrane proteins

Fundamental biological processes such as cell-cell communication, signal transduction, molecular transport and energy conversion are performed by membrane proteins. These important proteins are studied best in their native environment, the lipid bilayer. The atomic force microscope (AFM) is the instrument of choice to determine the native surface structure, supramolecular organization, conformational changes and dynamics of membrane-embedded proteins under near-physiological conditions. In addition, membrane proteins are imaged at subnanometer resolution and at the single molecule level with the AFM. This review highlights the major advances and results achieved on reconstituted membrane proteins and native membranes as well as the recent developments of the AFM for imaging.

Angiotensin Ii As A General Growth Factor

Angiotensin II (Ang II), a key protein in the renin-angiotensin system, can induce cardiac hypertrophy through an intracrine system as well as affect gene transcription. The receptor to Ang II responsible for this effect, AT1, has been localized to the nucleus of cell types in addition to cardiomyocytes. In this study, we induced expression of Ang II in MC3T3 osteoblasts and K7M2 osteosarcomas and measured changes in protein expression of Annexin V and matrix metalloproteinase 2 (MMP2), proteins identified previously through mass spectrometry analysis as being regulated by Ang II. Annexin V is downregulated in both immortalized murine bone (MC3T3) cells and in cancerous immortalized murine (K7M2) cells induced to express Ang II. MC3T3 cells which express Ang II show a downregulation of MMP2 expression, but Ang II-expressing K7M2 cells show an upregulation of MMP2. The differential regulation of MMP2 between the cancerous cells and noncancerous cells implicates a role for Ang in in tumor metastasis, as MMP2 is a metastatic protein. Annexin V is used as a marker for apoptosis, but nothing is known of the function of the endogenous protein. That Annexin V is potentially regulated by Ang II provides more information with which to characterize the protein and could suggest a function for Annexin V as part of a signal transduction pathway inside of the cell.

EFFECTS OF PRENATAL DEXAMETHASONE ON HIPOPCAMPAL SEROTONIN 1A RECEPTORS IN ADULT MALE RATS

The main activation route for the stress response is the hypothalamo-pituitaryadrenal axis (HPA) and the sympatho-adrenomedullary system. The HPA axis is a neuroendocrine feedback loop mediated by an array of tissue specific hormones, receptors and neurotransmitters that regulate glucocorticoid (GC) release. GCs are steroidal hormones produced by the adrenal glands and are key players in a negativefeedback loop controlling HPA activity. They influence the HPA axis through glucocorticoid receptors in the hypothalamus and pituitary and through both glucocorticoid (GR) and mineralcorticoid receptors (MR) that are co-localized in the hippocampus. Repeated or chronic stress exerts a negative influence on these HPA axis regulatory sites and contributes to potentially pathological conditions, especially during early development. For example, chronic stress promotes increased maternal adrenal gland secretion of glucocortiocoid, leading to abnormally high concentrations of GC inthe fetal environment. The timing and maturation of the HPA axis relative to birth is highly species specific and is closely linked to landmarks in fetal development. In rats this development of the HPA axis takes place in utero and continues even shortly after birth. It is likely that the maternal endocrine environment will affect fetal development during this critical time point and may alter the overall set point for the expression ofgenes and their protein products that mediate fetal HPA axis function. Dexamethasone (DEX) is a synthetic glucocorticoid (sGC) and is a consensus treatment in preterm pregnancies used to expedite fetal lung development. However it has been shown that DEX causes long term physiological and behavioral disorders in prenatally-exposed laboratory animals. Previous studies have also shown that it alters the MR: GR receptor ratio in the hippocampus. Taking into consideration corticosteroid regulation of serotonin receptors, especially 5HT1A receptors and their putative interaction with glucocorticoid receptors in the hippocampus, we hypothesized that prenatal DEX exposure would lead to changes in the expression and function of 5HT1A receptors in the hippocampus. We administered DEX to rat dams during the last trimester of gestation and investigated the changes in these receptors in the adult rat offspring. Radioligand receptor binding assays were used to study hippocampal 5HT1A receptor binding affinity and number. Our results demonstrate that hippocampal 5HT1A receptors are increased in the DEX animalscompared with controls by 36%, with no change in binding affinity. The efficiency of ligand-induced receptor signal transduction via G-protein activation was also studied using [35S]GTPγS incorporation assay. Using this technique, we showed that there was no significant difference in the maximum ligand mediated stimulation (Emax) of 5HT1Areceptors between control and dex exposed animals. However, the intracellular signalling efficiency of hippocampal 5HT1A receptors was diminished, since a significant increase in EC50 values was obtained with the dex exposed group showing a value 51% higherEC50 than controls. Taken together these data illustrate a considerable change in the 5HT1A component of the serotonergic system following prenatal DEX exposure.

Structure-Function Relations in the PI-PLC/GDPD Enzyme Superfamily

Phosphatidylinositol-specific phospholipases C (PI-PLC) are known to participate in many eukaryotic signal transduction pathways and act as virulence factors in lower organisms. Glycerophosphoryl diester phosphodiesterase (GDPD) enzymes are involved in phosphate homeostasis and phospholipid catabolism for energy production. Streptomyces antibioticus phosphatidylinositol-specific phospholipase C (SaPLC1) is a 38 kDa enzyme that displays characteristics of both enzyme superfamilies, representing an evolutionary link between these divergent enzyme classes. SaPLC1 also boasts a unique catalytic mechanism that involves a trans 1,6-cyclic inositol phosphate intermediate instead of the typical cis 1,2-cyclic inositol phosphate. The mechanism by which this occurs is still unclear. To attack this problem, we established a wide mutagenesis scan of the active site and measured activities of alanine mutants. A chemical rescue assay was developed to verify that the activity loss was due to the removal of the functional role of the mutated residue. 31P-NMR was employed in characterizing and quantifying intermediates in mutants that slowed the reaction sufficiently. We found that the H37A and H76A mutations support the hypothesis that these structurally conserved residues are also conserved in terms of their catalytic roles. H37 was found to be the general base (GB), while H76 plays the role of general acid (GA). K131 was identified as a semi-conserved key positive charge donor found at the entrance of the active site. By elucidating the SaPLC1 mechanism in relation to its active site architecture, we have increased our understanding of the structure-function relations that support catalysis in the PI-PLC/GDPD superfamily. These findings provide groundwork for in vivo studies of SaPLC1 function and its possible role in novel signaling or metabolism in Streptomyces.

β-Arrestin2 influences the response to methadone in opioid-dependent patients

β-Arrestin2 (ARRB2) is a component of the G-protein-coupled receptor complex and is involved in μ-opioid and dopamine D(2) receptor signaling, two central processes in methadone signal transduction. We analyzed 238 patients in methadone maintenance treatment (MMT) and identified a haplotype block (rs34230287, rs3786047, rs1045280 and rs2036657) spanning almost the entire ARRB2 locus. Although none of these single nucleotide polymorphisms (SNPs) leads to a change in amino-acid sequence, we found that for all the SNPs analyzed, with exception of rs34230287, homozygosity for the variant allele confers a nonresponding phenotype (n=73; rs1045280C and rs2036657G: OR=3.1, 95% CI=1.5-6.3, P=0.004; rs3786047A: OR=2.5, 95% CI=1.2-5.1, P=0.02) also illustrated by a 12-fold shorter period of negative urine screening (P=0.01). The ARRB2 genotype may thus contribute to the interindividual variability in the response to MMT and help to predict response to treatment.

Identification of a highly conserved valine-glycine-phenylalanine amino acid triplet required for HIV-1 Nef function

Background The Nef protein of HIV facilitates virus replication and disease progression in infected patients. This role as pathogenesis factor depends on several genetically separable Nef functions that are mediated by interactions of highly conserved protein-protein interaction motifs with different host cell proteins. By studying the functionality of a series of nef alleles from clinical isolates, we identified a dysfunctional HIV group O Nef in which a highly conserved valine-glycine-phenylalanine (VGF) region, which links a preceding acidic cluster with the following proline-rich motif into an amphipathic surface was deleted. In this study, we aimed to study the functional importance of this VGF region. Results The dysfunctional HIV group O8 nef allele was restored to the consensus sequence, and mutants of canonical (NL4.3, NA-7, SF2) and non-canonical (B2 and C1422) HIV-1 group M nef alleles were generated in which the amino acids of the VGF region were changed into alanines (VGF→AAA) and tested for their capacity to interfere with surface receptor trafficking, signal transduction and enhancement of viral replication and infectivity. We found the VGF motif, and each individual amino acid of this motif, to be critical for downregulation of MHC-I and CXCR4. Moreover, Nef’s association with the cellular p21-activated kinase 2 (PAK2), the resulting deregulation of cofilin and inhibition of host cell actin remodeling, and targeting of Lck kinase to the trans-golgi-network (TGN) were affected as well. Of particular interest, VGF integrity was essential for Nef-mediated enhancement of HIV virion infectivity and HIV replication in peripheral blood lymphocytes. For targeting of Lck kinase to the TGN and viral infectivity, especially the phenylalanine of the triplet was essential. At the molecular level, the VGF motif was required for the physical interaction of the adjacent proline-rich motif with Hck. Conclusion Based on these findings, we propose that this highly conserved three amino acid VGF motif together with the acidic cluster and the proline-rich motif form a previously unrecognized amphipathic surface on Nef. This surface appears to be essential for the majority of Nef functions and thus represents a prime target for the pharmacological inhibition of Nef.

Differential modulation of ROS signals and other mitochondrial parameters by the antioxidants MitoQ, resveratrol and curcumin in human adipocytes

Abstract Mitochondrial reactive oxygen species (ROS) have been demonstrated to play an important role as signaling and regulating molecules in human adipocytes. In order to evaluate the differential modulating roles of antioxidants, we treated human adipocytes differentiated from human bone marrow-derived mesenchymal stem cells with MitoQ, resveratrol and curcumin. The effects on ROS, viability, mitochondrial respiration and intracellular ATP levels were examined. MitoQ lowered both oxidizing and reducing ROS. Resveratrol decreased reducing and curcumin oxidizing radicals only. All three substances slightly decreased state III respiration immediately after addition. After 24 h of treatment, MitoQ inhibited both basal and uncoupled oxygen consumption, whereas curcumin and resveratrol had no effect. Intracellular ATP levels were not altered. This demonstrates that MitoQ, resveratrol and curcumin exert potent modulating effects on ROS signaling in human adipocyte with marginal effects on metabolic parameters.