Disturbed Epidermal Structure in Mice with Temporally Controlled Fatp4 Deficiency

So far, little is known about the physiological role of fatty acid transport protein 4 (Fatp4, Slc27a4). Mice with a targeted disruption of the Fatp4 gene display features of a human neonatally lethal restrictive dermopathy with a hyperproliferative hyperkeratosis, a disturbed epidermal barrier, a flat dermal–epidermal junction, a reduced number of pilo-sebaceous structures, and a compact dermis, demonstrating that Fatp4 is necessary for the formation of the epidermal barrier. Because Fatp4 is widely expressed, it is unclear whether intrinsic Fatp4 deficiency in the epidermis alone can cause changes in the epidermal structure or whether the abnormalities observed are secondary to the loss of Fatp4 in other organs. To evaluate the functional role of Fatp4 in the skin, we generated a mouse line with Fatp4 deficiency inducible in the epidermis. Mice with epidermal keratinocyte-specific Fatp4 deficiency developed a hyperproliferative hyperkeratosis with a disturbed epidermal barrier. These changes resemble the histological abnormalities in the epidermis of newborn mice with total Fatp4 deficiency. We conclude that Fatp4 in epidermal keratinocytes is essential for the maintenance of a normal epidermal structure

COX-2 mRNA Expression is Significantly Increased in Acid-exposed Compared to Nonexposed Squamous Epithelium in Gastroesophageal Reflux Disease

Background: Little is known about the role of cyclooxygenase (COX)-2 in gastroesophageal reflux disease (GERD) and the development of Barrett's metaplasia. The objectives of this study were to further analyze COX-2 mRNA expression in patients with GERD compared to Barrett's esophagus (BE) and Barrett's cancer (BC). Methods: Tissue samples from 110 patients with GERD (n = 43), BE (n = 20), and BC (n = 47) were obtained in routine upper GI endoscopy. Expression levels of COX-2 were measured by quantitative real-time reverse trancriptase polymerase chain reaction (RT-PCR). Also, 24-h pH monitoring was performed in all patients of the GERD study group and the DeMeester composite score was used to match COX-2 mRNA expression with the severity of acid exposure in the lower esophagus. Results: COX-2 mRNA is progressively upregulated within the metaplasia-dysplasia-adenocarcinoma (MDA) sequence (p = 0.001). COX-2 levels of the squamous epithelium in the distal esophagus from patients with GERD and a pathologic mean DeMeester score (>14.72) were significantly higher than in patients with normal DeMeester scores (p = 0.01). Conclusion: In summary our findings suggest that alterations in COX-2 mRNA expression occur independently of endoscopic or histologic signs of GERD in the acid-exposed squamous epithelium of the distal esophagus. However, this early COX-2 increase in GERD is further upregulated within the MDA sequence for yet unknown reason

Coordinating Role of RXR alpha in Downregulating Hepatic Detoxification during Inflammation Revealed by Fuzzy-Logic Modeling

During various inflammatory processes circulating cytokines including IL-6, IL-1 beta, and TNF alpha elicit a broad and clinically relevant impairment of hepatic detoxification that is based on the simultaneous downregulation of many drug metabolizing enzymes and transporter genes. To address the question whether a common mechanism is involved we treated human primary hepatocytes with IL-6, the major mediator of the acute phase response in liver, and characterized acute phase and detoxification responses in quantitative gene expression and (phospho-)proteomics data sets. Selective inhibitors were used to disentangle the roles of JAK/STAT, MAPK, and PI3K signaling pathways. A prior knowledge-based fuzzy logic model comprising signal transduction and gene regulation was established and trained with perturbation-derived gene expression data from five hepatocyte donors. Our model suggests a greater role of MAPK/PI3K compared to JAK/STAT with the orphan nuclear receptor RXR alpha playing a central role in mediating transcriptional downregulation. Validation experiments revealed a striking similarity of RXRa gene silencing versus IL-6 induced negative gene regulation (r(s) = 0.79;P<0.0001). These results concur with RXRa functioning as obligatory heterodimerization partner for several nuclear receptors that regulate drug and lipid metabolism

Differential roles of nitric oxide synthase isozymes in cardiotoxicity and mortality following chronic doxorubicin treatment in mice

The roles of individual nitric oxide synthases (NOS) in anthracycline-related cardiotoxicity are not completely understood. We investigated the effects of a chronic treatment with doxorubicin (DOX) on knockouts of the individual NOS isozymes and on transgenic mice with myocardial overexpression of eNOS. Fractional shortening (FS) was reduced in untreated homozygous nNOS and iNOS knockouts as well as in eNOS transgenics. DOX-induced FS decrease in wild-type mice was attenuated only in eNOS knockouts, which were found to overexpress nNOS. No worsening of contractility was observed in DOX-treated eNOS transgenics and iNOS knockouts. Although the surviving DOX-treated nNOS knockouts exhibited no further impairment in contractility, most (70%) animals died within 7 weeks after treatment onset. In comparison to untreated wild-type hearts, the nitric oxide (NO) level was lower in hearts from DOX-treated wild-type mice and in all three untreated knockouts. DOX treatment had no effect on NO in the knockouts. These data indicate differential roles of the individual NOS in DOX-induced cardiotoxicity. Protection against DOX effects conferred by eNOS deletion may be mediated by a compensatory overexpression of nNOS. NOS inhibition-based prevention of anthracycline-induced cardiotoxicity should be eNOS-selective, simultaneously avoiding inhibiting nNOS

Towards long-term standardised carbon and greenhouse gas observations for monitoring Europe's terrestrial ecosystems : a review

Research infrastructures play a key role in launching a new generation of integrated long-term, geographically distributed observation programmes designed to monitor climate change, better understand its impacts on global ecosystems, and evaluate possible mitigation and adaptation strategies. The pan-European Integrated Carbon Observation System combines carbon and greenhouse gas (GHG; CO2, CH4, N2O, H2O) observations within the atmosphere, terrestrial ecosystems and oceans. High-precision measurements are obtained using standardised methodologies, are centrally processed and openly available in a traceable and verifiable fashion in combination with detailed metadata. The Integrated Carbon Observation System ecosystem station network aims to sample climate and land-cover variability across Europe. In addition to GHG flux measurements, a large set of complementary data (including management practices, vegetation and soil characteristics) is collected to support the interpretation, spatial upscaling and modelling of observed ecosystem carbon and GHG dynamics. The applied sampling design was developed and formulated in protocols by the scientific community, representing a trade-off between an ideal dataset and practical feasibility. The use of open-access, high-quality and multi-level data products by different user communities is crucial for the Integrated Carbon Observation System in order to achieve its scientific potential and societal value.Peer reviewe

Genome-wide association and functional follow-up reveals new loci for kidney function

Chronic kidney disease (CKD) is an important public health problem with a genetic component. We performed genome-wide association studies in up to 130,600 European ancestry participants overall, and stratified for key CKD risk factors. We uncovered 6 new loci in association with estimated glomerular filtration rate (eGFR), the primary clinical measure of CKD, in or near MPPED2, DDX1, SLC47A1, CDK12, CASP9, and INO80. Morpholino knockdown of mpped2 and casp9 in zebrafish embryos revealed podocyte and tubular abnormalities with altered dextran clearance, suggesting a role for these genes in renal function. By providing new insights into genes that regulate renal function, these results could further our understanding of the pathogenesis of CKD

New genetic loci link adipose and insulin biology to body fat distribution.

Body fat distribution is a heritable trait and a well-established predictor of adverse metabolic outcomes, independent of overall adiposity. To increase our understanding of the genetic basis of body fat distribution and its molecular links to cardiometabolic traits, here we conduct genome-wide association meta-analyses of traits related to waist and hip circumferences in up to 224,459 individuals. We identify 49 loci (33 new) associated with waist-to-hip ratio adjusted for body mass index (BMI), and an additional 19 loci newly associated with related waist and hip circumference measures (P < 5 × 10(-8)). In total, 20 of the 49 waist-to-hip ratio adjusted for BMI loci show significant sexual dimorphism, 19 of which display a stronger effect in women. The identified loci were enriched for genes expressed in adipose tissue and for putative regulatory elements in adipocytes. Pathway analyses implicated adipogenesis, angiogenesis, transcriptional regulation and insulin resistance as processes affecting fat distribution, providing insight into potential pathophysiological mechanisms

Structural and biochemical investigations of prenyltransferases and amide synthetases from the ergot alkaloid and aminocoumarin antibiotic biosynthesis

Die Dimethylallyltryptophansynthase (DMATS) katalysiert den ersten Schritt der Ergotalkaloid-Biosynthese und prenyliert dabei L-Trp am C-4 des Indolrings mit Dimethylallylpyrophosphat (DMAPP) als Prenyldonor. Eine Vielzahl von Orthologen der DMATS wurde in Genomen von verschiedenen Pilzen nachgewiesen, aber von keinem der von diesen Genen codierten Enzyme konnte in der Vergangenheit die 3D Struktur ermittelt werden. Nun konnte zum ersten Mal die Röntgenstruktur einer DMATS aufgeklärt werden. Die Struktur von FgaPT2, der DMATS aus Aspergillus fumigatus, wurde mit einer Auflösung von 1,76 Å bestimmt. Die Kristallform gehört zur Raumgruppe P212121 und enthält zwei Moleküle in der asymmetrischen Einheit. Die 3D Struktur zeigt eine aus alpha,beta-Einheiten bestehende barrel-Struktur, die kürzlich erstmals in einem bakteriellen Enzym (NphB) entdeckt und Prenyltransferase (PT)-barrel genannt wurde. Obwohl NphB auch die Prenylierung eines aromatischen Substrats katalysiert, besitzt FgaPT2 zu diesem Enzym keine Sequenzähnlichkeit. Die Struktur von FgaPT2 mit dem gebundenen Substrat L-Trp und dem gebundenen nicht-hydrolysierbaren Substratanalogon DMASPP konnte mit einer Auflösung von 2,2 Å ermittelt werden. Auf der Grundlage dieser Struktur wurde ein Modell zur Beschreibung einer enzymatisch katalysierten Friedel-Crafts-Alkylierung entwickelt, das auch Erklärungen für die strenge Regiospezifität der DMATS-Reaktion am C-4 und die ungewöhnliche Mg2+-Unabhängigkeit des Enzyms liefert. Die Funktion einzelner Aminosäuren wurde herausgearbeitet und ein detaillierter Reaktionsmechanismus vorgeschlagen. Die Prenyltransferase CloQ aus Streptomyces roseochromogenes katalysiert eine ähnliche Reaktion wie FgaPT2 und zeigt ähnliche biochemische Eigenschaften, aber keine Sequenzähnlichkeit zu FgaPT2. CloQ katalysiert die Prenylierung von 4-Hydroxyphenylpyruvat (4-HPP) mit DMAPP als Prenyldonor in der Biosynthese des Antibiotikums Clorobiocin. Es wurden vier Strukturen von CloQ gelöst: CloQ mit gebundenem 4-HPP (2,5 Å), CloQ ohne Substrat (3,1 Å), CloQ mit der Mutation C215S (1,9 Å) und CloQ mit der Mutation R66S und gebundenem 4-HPP (2,2 Å). Alle Kristallformen gehören zur Raumgruppe I4122. Die Raumstruktur von CloQ entspricht dem oben genannten PT-barrel. Trotz intensiver Versuche gelang es nicht, das isoprenoide Substrat DMAPP in der Struktur zu zeigen. Jedoch konnte durch Sequenz- und Strukturvergleiche mit FgaPT2 und NphB eine hypothetische Bindestelle von DMAPP vorgeschlagen und durch Mutationsexperimente verifiziert werden. Die in der Röntgenstruktur eindeutig erkennbare kovalente Bindung des aromatischen Substrats mit C215 in Form einer Thiohemiketalbindung ist für die katalytische Reaktion nicht relevant, was ebenfalls durch Mutationen belegt wurde. Durch Vergleich der CloQ-Struktur mit den Strukturen von FgaPT2 und NphB konnte die Funktion mehrerer Aminosäuren in CloQ definiert und durch Mutationsexperimente bestätigt werden. Ein Reaktionsmechanismus, der ähnlich verläuft wie bei FgaPT2, konnte dadurch vorgeschlagen werden. Die in der Literatur diskutierte Promiskuität der aromatischen Prenyltransferasen wurde auch für CloQ durch Umsetzung der artifiziellen Substrate L-Tyr (Km = 680 my M), p-Cumarsäure (Km = 874 my M) und Geranylpyrophosphat (Km = 307 my M) beobachtet. Die kinetischen Parameter von CloQ mit den genuinen Substraten zeigen jedoch ganz klar eine Präferenz für die genuinen Substrate (Km 4-HPP = 16 my M, Km DMAPP = 25 my M). Die katalytische Effizienz für deren Umsetzung ist im Vergleich zu den artifiziellen Substraten 1000-mal höher. Die Amidsynthetase NovL, ein Schlüsselenzym der Biosynthese des Antibiotikums Novobiocin, katalysiert sowohl die Aktivierung seines Säuresubstrats durch eine Adenylierung als auch den anschließenden Acyltransfer auf die Aminogruppe des Aminocoumarinrings. NovL wurde kristallisiert und ein Datensatz mit einer Auflösung von 2,6 Å aufgenommen. Die Bestimmung der Proteinphasen war aufgrund der Zwillingsbildung der Kristalle jedoch bislang nicht möglich. Parallel zur strukturbiologischen Untersuchung von NovL wurden biochemische Untersuchungen an den Amidsynthetasen NovL und CouL durchgeführt. Zur Untersuchung der für die Substraterkennung verantwortlichen Sequenzbereiche wurden zehn verschiedene Chimäre aus diesen Enzymen hergestellt und deren Aktivität mit den genuinen Substraten (3-Dimethylallyl-4-hydroxybenzoes?ure und 3-Methylpyrrol-2,4-dicarbonsäure) und einem artifiziellen Substrat (3-Propyl-4-hydroxybenzoesäure) untersucht. Die für die Substraterkennung verantwortlichen Bereiche liegen bei NovL (CouL) zwischen den Aminosäuren 215 bis 412 (217 bis 414). Dies wird auch durch einen Sequenzvergleich der beiden Amidsynthetasen mit zwei Adenylierungsdomänen (DhbE und PheA) von nicht-ribosomalen Peptidsynthetasen gestützt. Mit Hilfe der Sequenzvergleiche können einzelne Aminosäuren vorgeschlagen werden, die wahrscheinlich von besonderer Bedeutung für die Substratspezifität sind.The dimethylallyl tryptophan synthase (DMATS) catalyzes the first committed step of ergot alkaloid biosynthesis, i.e. the prenylation of L-tryptophan at C-4 of the indole moiety with the isoprenoid substrate dimethylallyl diphosphate. Orthologs of DMATS are found in many fungal genomes, but no 3D structure of these enzymes has been determined yet. In this thesis the first x-ray structure of a DMATS is presented. The structure of FgaPT2, the DMATS from Aspergillus fumigatus, was determined at a resolution of 1.76 Å. The crystals belong to the space group P212121 and contain two molecules in the asymmetric unit. The 3D structure belongs to a rare beta/alpha barrel fold, called prenyltransferase (PT) barrel, which was recently discovered in the bacterial enzyme NphB. NphB also catalyzes the prenylation of an aromatic substrate but does not show detectable primary sequence similarity to DMATS. The structure of FgaPT2 complexed with the substrate L-tryptophan and with the non hydrolysable DMAPP-analogue DMASPP was determined to a resolution of 2.2 Å. On basis of the structure a detailed reaction mechanism was suggested for this enzyme-catalyzed Friedel-Crafts-alkylation which explains the strict regiospecificity of the DMATS reaction for prenylation at C-4 and the unusual independence of Mg2+. The function of several residues was investigated by site-directed mutagenesis. The prenyltransferase CloQ from the clorobiocin biosynthetic pathway of Streptomyces roseochromogenes catalyzes, like FgaPT2, a prenylation of an aromatic substrate. The two enzymes show similar biochemical properties but do not show detectable primary sequence similarity. CloQ catalyzes the attachment of a dimethylallyl moiety to 4-hydroxyphenylpyruvate (4-HPP). The structure of wild-type CloQ, with 4-HPP bound, was determined at 2.2 Å resolution. Three further structures were determined, the wild-type protein in the absence of substrates (3.1 Å), the C215S mutant (1.9 Å) and the R66S mutant, with 4-HPP bound (2.2 Å). All crystals belong to the space group I4122. The CloQ structure also shows the PT barrel fold mentioned above. Despite exhaustive attempts it was not possible to obtain a structure of CloQ complexed with the isoprenoid substrate DMAPP. A structure-based sequence alignment and superpositions of CloQ with the ligand-bound structures of NphB and FgaPT2 resulted in a plausible model of the binding site of DMAPP. This was confirmed by site-directed mutagenesis. 4-HPP forms a covalent link to C215 to give a thiohemiketal, what is evidenced by the continuous electron density between the protein and the ligand. However, site-directed mutagenesis experiments showed that this bond is not relevant for catalytic activity. By superposition of CloQ with the ligand-bound structures of NphB and FgaPT2, a hypothesis for the function of several amino acid residues in CloQ was generated and verified by site-directed mutagenesis. A reaction mechanism similar to FgaPT2 was suggested. The previously described promiscuity of the aromatic prenyltransferases was also confirmed for CloQ with the artificial substrates L-tyrosine (Km = 680 my M), p-coumaric acid (Km = 874 my M), and geranyl pyrophosphate (Km = 307 my M). However, the determination of the kinetic parameters of the genuine substrates demonstrated a clear preference of CloQ to the genuine substrates (Km 4-HPP = 16 my M, Km DMAPP = 25 my M). The catalytic efficiency for the genuine substrate is 1000 fold higher than for the artificial substrates. The amide synthetase NovL is a key enzyme of the biosynthesis of the antibiotic novobiocin. NovL catalyzes the activation of its acyl substrate as acyl adenylate and the subsequent transfer of the acyl group to the amino group of the aminocoumarin ring. In this thesis, NovL was crystallized and a data set was collected to a resolution of 2.6 Å. Due to the twinning of the NovL crystals, the protein phases could not be determined yet. In addition to the structural investigation of NovL, NovL and the amide synthetase CouL were biochemically investigated. Ten different chimera of NovL and CouL were generated, the activity was determined with the genuine substrates (3-dimethylallyl-4-hydroxybenzoic acid and 3-methylpyrrole-2,4-dicarboxylic acid) and with one artificial substrate (3-propyl-4-hydroxybenzoic acid) to investigate the relevance of several regions of the amino acid sequences for substrate recognition. The important amino acids for substrate recognition are located in NovL (CouL) in the region comprising amino acids 215 to 412 (217 to 414). This was confirmed by the sequence alignment of both amide synthetases with two adenylation domains (DhbE and PheA) of nonribosomal peptide synthetases. Due to this alignment several amino acids were suggested to play an important role in substrate recognition