Evolutionary aspects in evaluating mutations in the melanocortin 4 receptor

More than 70 missense mutations have been identified in the human melanocortin 4 receptor (MC4R), and many of them have been associated with obesity. In a number of cases, the causal link between mutations in MC4R and obesity is controversially discussed. Here, we mined evolution as an additional source of structural information that may help to evaluate the functional relevance of naturally occurring variations in MC4R. The sequence information of more than 60 MC4R orthologs enabled us to identify residues that are important for maintaining receptor function. More than 90% of all inactivating mutations found in obese patients were located at amino acid positions that are highly conserved during 450 million years of MC4R evolution in vertebrates. However, for a reasonable number of MC4R variants, we found no correlation between structural conservation of the mutated position and the reported functional consequence. By re-evaluating selected mutations in the MC4R, we demonstrate the usefulness of combining functional and evolutionary approaches

Differential cellular metabolite alterations in HaCaT cells caused by exposure to the aryl hydrocarbon receptor-binding polycyclic aromatic hydrocarbons chrysene, benzo[a]pyrene and dibenzo[a,l]pyrene

Polycyclic aromatic hydrocarbons (PAHs) are ubiquitous in the human environment. Since they are present in crude oilfractions used for the production of rubber and plastics, consumers may come into direct dermal contacts with these compounds (e.g., via tool handles) on a daily basis. Some individual PAHs are identified as genotoxic mutagens thereby prompting particular toxicological and environmental concern. Among this group, benzo[a]pyrene (BAP) constitutes a model carcinogen which is also used as reference compound for risk assessment purposes. It acts as a strong agonist of the aryl hydrocarbon receptor (AHR) and becomes metabolically activated toward mutagenic and carcinogenic intermediates by cytochrome P450-dependent monooxygenases (CYPs). While BAP has been exhaustively characterized with regard to its toxicological properties, there is much less information available for other PAHs. We treated an AHR-proficient immortal human keratinocyte cell line (i.e., HaCaT) with three selected PAHs: BAP, chrysene (CRY) and dibenzo[a,l]pyrene (DALP). Compound-mediated alterations of endogenous metabolites were investigated by an LC–MS/MS-based targeted approach. To examine AHR-dependent changes of the measured metabolites, AHR-deficient HaCaT knockdown cells (AHR-KD) were used for comparison. Our results reveal that 24 metabolites are sufficient to separate the PAH-exposed cells from untreated controls by application of a multivariate model. Alterations in the metabolomics profiles caused by each PAH show influences on the energy and lipid metabolism of the cells indicating reduced tricarboxylic acid (TCA) cycle activity and β-oxidation. Up-regulation of sphingomyelin levels after exposure to BAP and DALP point to pro-apoptotic processes caused by these two potent PAHs. Our results suggest that in vitro metabolomics can serve as tool to develop bioassays for application in hazard assessment. Keywords: Polycyclic aromatic hydrocarbons, Metabolomics, Aryl hydrocarbon receptor, Keratinocyte

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

Recommendations on basic requirements for intensive care units: structural and organizational aspects

OBJECTIVE: To provide guidance and recommendations for the planning or renovation of intensive care units (ICUs) with respect to the specific characteristics relevant to organizational and structural aspects of intensive care medicine. METHODOLOGY: The Working Group on Quality Improvement (WGQI) of the European Society of Intensive Care Medicine (ESICM) identified the basic requirements for ICUs by a comprehensive literature search and an iterative process with several rounds of consensus finding with the participation of 47 intensive care physicians from 23 countries. The starting point of this process was an ESICM recommendation published in 1997 with the need for an updated version. RESULTS: The document consists of operational guidelines and design recommendations for ICUs. In the first part it covers the definition and objectives of an ICU, functional criteria, activity criteria, and the management of equipment. The second part deals with recommendations with respect to the planning process, floorplan and connections, accommodation, fire safety, central services, and the necessary communication systems. CONCLUSION: This document provides a detailed framework for the planning or renovation of ICUs based on a multinational consensus within the ESIC

Molekulare Charakterisierung an der hypothalamischen Appetitregulation beteiligter Rezeptoren

Das Körpergewicht und die Nahrungsaufnahme werden unter anderem vom Hypothalamus reguliert. Dort werden Hormonelle Signale der Peripherie und neuronale Signale integriert. Die G-Protein gekoppelten Melanocortinrezeptoren 3 und 4 (MC3R und MC4R) werden von ihren Agonisten, den Melanocortinen aktiviert und durch den inversen Agonisten/Antagonisten Agouti-Related Peptide (AgRP) inaktiviert. Als weiterer Downstream-Mediatoren der MC4R-Aktivierung wurden kürzlich Brain Derived Neurotrophic Factor (BDNF) und dessen Rezeptor TrkB (Tropomyosin-Related –Kinase) identifiziert. Mutationen im MC4R gelten als häufigste monogenetische Ursache für Adipositas. Da viele dieser Mutationen aber in vitro funktionell nicht relevant sind, wurde ein Amosäurevergleich von orthologen MC4R aus 70 verschiedenen Spezies erstellt. Funktionsverlustmutationen waren häufiger an koservierten Positionen, während Mutationen ohne Effekt überwiegend an schwach konservierten Positionen zu finden waren. Funktionelle Charakterisierung der von in Mausmodellen identifizierten Punktmutationen I194F und Y302C ergaben eine gute in-vivo/in-vitro Korrelation. Desweiteren wurden in der Normalbevölkerung in normalgewichtigen Personen identifizierte MC4R-Punktmutationen funktionell charakterisiert. Die Mutationen R7C, A70T, T112K, Q156R, M200V, V166I und R236H hatten keinen Effekt auf die Rezeptorfunktion, die H158R. Mutation zeigte eine hohe Basalaktivität, die aber durch AgRP erniedrigt werden konnte. Die in adipösen Patienten gefundenen Mutationen S136F und S139R wiesen einen kompletten Funktionsverlust auf, erstere verursachte zudem sogar einen dominant-negativen Effekt bei Koexpression mit dem Wildtyprezeptor. Für den MC3R wurde das zum Translationsstart bevorzugte Startcodon identifiziert. Für die Rezeptortyrosinkinase TrkB konnte in Hefe-2-Hybridscreens der neue Interaktionspartner Sept3 identifiziert werden. Dieses Protein bindet phosphorylierungsunabhängig an die intrazelluläre Juxtamembrandomäne.Bodyweight and food intake are regulated by the hypothalamus which integrates peripheral hormonal and neural signals. The G-protein-coupled melanocortin-receptors 3 and 4 (MC3R and MC4R) are activated by melanocortins or inhibited by agouti-related pepetide (AgRP) and signal via the cAMP pathway. Brain-derived neurotrophic Factor (BDNF) was recently shown to signal downstream the MC4R via its receptor TrkB (tropomyosin-related kinase). Mutations in the MC4R are the most common cause of monogenetic obesity. However, many of these mutations are not functionally relevant in vitro. Here, an amino acid alignment of orthologous MC4R from over 70 species was used to evaluate reported mutations. Loss-of-function mutations were predominantly located at highly conserved positions whereas mutations without effect were located at non-conserved positions. Functional characterization of MC4R point mutations I194F (partial loss of function) and Y302C (complete loss of function) identified in mouse models showed good in vitro/in vivo correlation. Furthermore mutations found in normal weight persons were characterized: R7C, A70T, T112K, Q156K, M200V, V166I and R236H had no effect on receptor function in vitro, whereas the H158R Mutation showed high constitutive activity, which however could be diminished by AgRP. The mutations S136F and S139F identified in obese patients were characterized as complete loss-of-function mutations, the former additionally caused a dominant-negative effect on wildtype MC4R in vitro. For the MC3R the preferred start-codon for initiation of translation was identified. For TrkB Sept3 could be identified as a new interaction partner in a yeast-2-hybrid screen. This Protein belonging to the septin family binds to the intracellular juxtamembrane domain of TrkB independent of phosphorylation of the Shc-binding site

Estrogenic Activity of Mineral Oil Aromatic Hydrocarbons Used in Printing Inks.

The majority of printing inks are based on mineral oils (MOs) which contain complex mixtures of saturated and aromatic hydrocarbons. Consumer exposure to these oils occurs either through direct skin contacts or, more frequently, as a result of MO migration into the contents of food packaging that was made from recycled newspaper. Despite this ubiquitous and frequent exposure little is known about the potential toxicological effects, particularly with regard to the aromatic MO fractions. From a toxicological point of view the huge amount of alkylated and unsubstituted compounds therein is reason for concern as they can harbor genotoxicants as well as potential endocrine disruptors. The aim of this study was to assess both the genotoxic and estrogenic potential of MOs used in printing inks. Mineral oils with various aromatic hydrocarbon contents were tested using a battery of in vitro assays selected to address various endpoints such as estrogen-dependent cell proliferation, activation of estrogen receptor α or transcriptional induction of estrogenic target genes. In addition, the comet assay has been applied to test for genotoxicity. Out of 15 MOs tested, 10 were found to potentially act as xenoestrogens. For most of the oils the effects were clearly triggered by constituents of the aromatic hydrocarbon fraction. From 5 oils tested in the comet assay, 2 showed slight genotoxicity. Altogether it appears that MOs used in printing inks are potential endocrine disruptors and should thus be assessed carefully to what extent they might contribute to the total estrogenic burden in humans

Estrogenic effects of MOs in the E-screen, the hERα-HeLa-9903 assay and induction of ER responsive transcripts as indicated.

<p>Proliferation assays with MCF-7 cells were performed subsequent to cellular stimulation with dispersions of 1 and 0.1 μl/ml MO (eq. to dil. of 1:1,000 and 1:10,000) or E2 as indicated (<b>A</b>). For the reporter gene assay hERα-HeLa-9903 cells were stimulated for 20 h with the indicated amounts of E2 or 1 μl/ml MO dispersed in medium, followed by cellular lysis and measurement of firefly luciferase activity (<b>B</b>). Transcriptional assays were following a 24-h exposure to 1 μl/ml MO or 10 nM E2, respectively (<b>C</b>). Data in all assays represent the mean ± SEM from at least three independent experiments. For the E-screen and the reporter gene assay data were corrected to accommodate the background of untreated cells and subjected to normalization using the effect of 1 nM E2. Likewise gene expression was normalized using <i>RPLP0</i> and the solvent control as references. Abbreviations: <i>PGR</i>, progesterone receptor (gene); <i>TFF1</i>, trefoil factor (gene); <i>GREB1</i>, estrogen-dependent growth regulator in breast cancer 1 (gene).</p

CYP induction and DNA damage caused by various MOs.

<p>Activation of AHR-dependent gene transcription in MCF-7 cells was measured following 24-h exposure to 1 μl/ml dispersed MO or 10 nM E2 (<b>A</b>). Data represent the mean ± SEM from at least three independent experiments. Gene expression levels were normalized to the reference gene <i>RPLP0</i> and the solvent control. For the comet assay (<b>B</b>), NHEKs were exposed to 1 μl/ml of dispersed MO, 3 μM benzo[<i>a</i>]pyrene (BP) or 20 μM methyl methanesulfonate (MMS) in presence of aphidicolin (APC). The resulting DNA damage was analyzed using an alkaline comet assay and quantified based on % DNA detected in the comet tails. Data represent the mean ± SEM of three independent experiments with cells from two individual donors resulting in 300 analyzed cells (* statistically significant (P<0.05) with respect to the APC/DMSO control as assessed by the Dunnett’s test). Abbreviations: <i>CYP1A1/1B1</i> (gene), cytochrome P450-dependent monooxygenase, family 1, subfamily A/B, polypeptide 1.</p