Acarbose hat als Inhibitor von Hydrolasen alpha-1,4-glykosidischer Bindungen medizinische Bedeutung. Das Acarbose-Biosynthese-Gencluster (acb) des grampositiven Produzenten Actinoplanes sp. wurde identifiziert und Genprodukte z. T. charakterisiert. Das Modell zum Acarbose-Metabolismus beschreibt einen Acarbosekreislauf, bei dem das Pseudotetrasaccharid als Carbophor fungiert. Das Molekül wird in das umgebende Medium abgegeben und durch das Zusammenwirken zweier extrazellulärer Enzyme nach Stärkehydrolyse mit einer unterschiedlichen Anzahl an Glukosemonomeren beladen. Nach dem vermuteten Re-Import über ein Bindeprotein-abhängiges ABC-Transportsystem AcbHFG stünde dem Organismus dann ein Gewinn an Glukosemolekülen zur Verfügung. Neben diesem Vorteil gegenüber Nahrungskonkurrenten im Habitat fungiert Acarbose ebenso als Hemmer der artfremden extrazellulären a-Amylasen. Die ökologische Funktion des Pseudotetrasaccharids wurde durch Untersuchungen zum Einfluss auf den Maltodextrin-Stoffwechsel von E. coli verifiziert und ausgeweitet. Es lässt sich ein ökonomisch sinnvolleres Konkurrenzverhalten von Actinoplanes sp. ableiten. Von den durch den Acarboseproduzenten selbst bereitgestellten Maltosacchariden aus Stärke profitieren artfremde Mikroorganismen nicht, da neben den Exoenzymen auch die Maltodextrin-Aufnahmesysteme in ihrer Funktion gehemmt sind. Außerdem wurde eine für Actinoplanes sp. geforderte Kapazität zur Aufnahme von Maltose und Maltodextrinen in vivo gefunden und in Transportexperimenten mit radioaktiv markierten Zuckern charakterisiert. Die Transportaktivität wird wahrscheinlich über zwei Bindeprotein-abhängige ABC-Importer mit multiplem Substratspektrum realisiert. Das ABC-Importsystem AcbHFG wurde heterolog in E. coli und S. lividans synthetisiert und z. T. erfolgreich gereinigt. In Substrat-Bindungsstudien konnte für das Bindeprotein AcbH eine Interaktion mit Acarbose und längerkettigen Derivaten, nicht jedoch mit Maltose/Maltodextrinen beobachtet werden. / Acarbose acts as an inhibitor of alpha-glucosidases and is therefore clinically used. The biosynthesis gene cluster (acb) was identified and partly characterized. The proposed model describes a pathway in which acarbose might function as a carbophor. The molecule is secreted into the medium where, after hydrolysis of starch, it is charged with additional glucose moieties. Re-uptake by a binding-protein dependent ABC importer AcbHFG would then result in a net gain of carbon and energy. Besides extracting glucose from the extracellular pool acarbose also acts as an inhibitor of alpha-amylases secreted by competitors in the natural environment. Prompted by the structural similarity between acarbose and maltotetraose, the effects of acarbose on the metabolism of maltose and maltodextrins in whole cells of E. coli and on individual components of the maltose / maltodextrin system were studied. The results demonstrate that acarbose is efficiently transported but not metabolized by E. coli due to its poor performance as a substrate of maltodextrin-degrading enzymes. Thus, besides acting as a carbophor acarbose also severely inhibits growth of competitors on maltodextrins. Actinoplanes using starch as carbon source should be able to import maltose and maltodextrins. Experiments with radioactive sugars indicate the action of two different binding-protein dependent ABC transport systems with a multiple substrate spectrum. Within the acb cluster a putative operon (acbHFG) encoding components of an ABC import system was found. To elucidate gene functions the products were overproduced in E. coli and S. lividans and some of the proteins were purified. Surface plasmon resonance analysis showed that the substrate binding protein AcbH binds acarbose and longer derivatives, but not maltose and maltodextrins.

The 5-HTT gene (SLC6A4) is regulated by a common polymorphism in the promoter region (5-HTTLPR), which has functional consequences. Two major alleles have been observed and shown to have differential transcriptional activity with the long (L) allele having greater gene expression than the short (S) allele. 5-HTTLPR appears to modulate depression, anxiety and personality traits such as neuroticism. Additionally, a significant influence of 5-HTTLPR genotype on amygdala reactivity in response to fearful stimuli has been reported. Moreover, 5-HTTLPR seems to impact on the role of stressful life events (SLEs) in the development of depression. An elevated risk of depression and suicidal behaviors has been found in carriers of at least one low expressing S allele who had experienced SLEs, suggesting a gene x environment interaction. However, a recent meta-analysis showed that several findings failed to replicate this finding. Since genetic polymorphisms of the dopaminergic and serotonergic neurotransmission interact at the molecular, analyses with another polymorphism of the dopaminergic system, the dopamine D4 receptor (DRD4) was included to consider these likely gene-gene interactions (epistasis). The aim of this series of studies was to investigate the role 5-HTTLPR and SLEs on the endocrine stress response in different age samples. While newborns have been examined by a heel prick, stress responses were provoked in children (8-12 yrs) and younger adults (19-31 yrs) and older adults (54-68 yrs.) with the Trier Social Stress Test (TSST). The Life History Calendar (LHC) and Life Events Questionnaire (LEQ) were used to acquire data on SLEs. While in newborns the S/S genotype showed a significantly higher acute endocrine stress response than L/L or S/L genotypes, no significant difference between genotype groups was found in children. In the younger adult sample, the genotype impacted on cortisol stress responsiveness was reversed. Adults carrying the more active L allele of the 5-HTTLPR polymorphism showed a significantly larger cortisol response to the TSST than individuals carrying at least one of the lower expressing S allele. In older adults, no significant difference between genotype groups was found. However, results point in the same direction with showing highest cortisol response in individuals with L/L genotype. These data suggest that the association between 5-HTTLPR and endocrine stress reactivity seems to alter across lifespan, more specific the effects of genotype turns around. In addition, a significant interaction effect of 5-HTTLPR and SLEs has been found in the sample of younger adults, i.e. that early SLE as well as a severe number SLEs across the entire lifespan seem to modulate the interaction between HPA axis activity and 5-HTTLPR genotype. Additionally, a DRD4 by 5-HTTLPR interaction emerged which point to independent and joint effects of these polymorphisms on stress responsivity with regard to the concept of genegene interaction.

No description available.

No description available.

Organische Anionen werden aus dem Blut in die proximalen Tubuluszellen durch Organische-Anionen-Transporter 1 und 3 (OAT1 und OAT3) aufgenommen. Die Aufnahme erfolgt im Austausch gegen Dicarboxylate. In dieser Dissertation wurde die Affinität von Dicarboxylaten gegenüber humanen OAT1 und OAT3 untersucht mit dem Ziel mehr Informationen über die Struktur der Transporter zu erhalten. Es sollten Unterschiede zwischen dem OAT1 und OAT3 ermittelt werden, besonders bezüglich deren Substratspezifität. Alle Transporter wurden stabil in HEK293-Zellen exprimiert. Extrazellulär wurden Dicarboxylate als Inhibitoren gegen die 3H-p-Aminohippurat-Aufnahme (OAT1) oder 3H-Östronsulfat-Aufnahme (OAT3) zugefügt. OAT1 zeigt die höchste Affinität gegenüber Glutarat (IC50 3,3 µM), α-Ketoglutarat (IC50 4,7 µM) und Adipat (IC50 6,2 µM), gefolgt von Pimelat (IC50 18,6 µM) und Suberat (IC50 19,3 µM). Die Affinität von OAT1 gegenüber Succinat und Fumarat war gering. Der OAT3 zeigte dieselbe Dicarboxylat-Selektivität mit etwa 13-mal höheren IC50-Werten verglichen mit dem OAT1. Die Daten charakterisieren α-Ketoglutarat als hochaffines Substrat für den OAT1 und den OAT3. Die Ergebnisse deuten auf eine ähnliche Molekülstruktur der Bindungsstellen von OAT1 und OAT3 hin.

The biological membrane surrounding the living cell provides a sealed barrier that tightly regulates the interactions with the outside environment. A large number of integral membrane proteins mediate these interactions and are involved in a wide variety of biological processes. An increasing number of studies have led to the conclusion that lipids provide more than a hydrophobic solvent for membrane proteins, and that interactions between lipids and proteins are required to allow protein function. ABC transporters are one of the most important family of membrane proteins. However, the importance of their lipidic environment is largely unknown. Only a few studies showed that their activity was dependent on the lipidic composition of the surrounding bilayer. The bacterial ABC transporter HorA was used as a model to probe the influence of the lipidic environment on that class of membrane proteins.<p><p> HorA is a multidrug transporter expressed in Lactobacillus brevis, a Gram-positive beer spoilage bacterium. It turned out that phosphatidylethanolamine (PE) was indispensable to maintain both the activity and the structural integrity of HorA.<p> Surprisingly, replacement of PE by the chemically related PC (phosphatidylcholine) did not led to the suppression of HorA activity, but to an unexpected phenotype. Whereas the cytoplasmic domains of HorA were still able to hydrolyze ATP, the membrane parts of the transporter were unable to use that energy to mediate substrate transport. Using several biophysical methods particularly adapted to the study of reconstituted systems, we showed that the structure of HorA is strongly altered by this lipid replacement. In particular, the structural organization of the transmembrane domains of the protein is strongly affected.<p> / Doctorat en Sciences agronomiques et ingénierie biologique / info:eu-repo/semantics/nonPublished

No description available.

L'obésité est une maladie multifactorielle qui constitue un facteur de risque de nombreuses pathologies, notamment les maladies cardiovasculaires, le diabète et les maladies neurodégénératives. Des études épidémiologiques récentes suggèrent un effet obésogène des contaminants environnementaux, mais peu d'informations sont disponibles sur leur effet potentiel sur le métabolisme des lipoprotéines hépatiques. L'objectif de cette étude était de déterminer l'effet de polluants environnementaux de la famille des hydrocarbures aromatiques polycycliques (HAP) sur trois récepteurs des lipoprotéines, le récepteur des LDL (LDL-R), le lipolysis-stimulated lipoprotein receptor (LSR) et le scavenger receptor B1 (SR-B1) ainsi que sur les ATP binding cassette transporter A1 (ABCA1) et G1 (ABCG1) en utilisant des modèles cellulaires et/ou animaux. Les études par immunoblots et immunofluorescence in vitro ont révélé que l'exposition de cellules Hepa1-6 au B[a]P diminue de manière significative les taux de protéine LSR, LDL-R et ABCA1, alors qu’aucune modification significative du taux et de l’activité du LSR n’a été observée lors d’une exposition au pyrène ou au phénanthrène. L’analyse en temps réel par PCR et les études avec la lactacystine ont révélé que cet effet était dû principalement à une augmentation de la dégradation par le protéasome plutôt qu’à une diminution de la transcription ou à une augmentation de la dégradation lysosomale. En outre, les ligand-blots ont révélé que les lipoprotéines exposées au B[a]P avaient une affinité réduite pour le LSR ou le LDL-R. Des souris C57Bl/6RJ ont été traitées par le B[a]P (0,5 mg / kg, i.p) toutes les 48 h pendant 15 jours. Le gain de poids observé est accompagné d’une augmentation des taux de triglycérides et de cholestérol plasmatiques, du taux de cholestérol hépatique, et d’une diminution du taux de LDL-R et ABCA1 chez animaux traités au B[a]P par rapport aux témoins. Les corrélations observées entre les taux de LSR et de LDL-R hépatiques chez les souris contrôle ne sont plus observées chez les souris traitées au B[a]P, ce qui suggère un dérèglement du métabolisme des lipoprotéines hépatiques. Ces résultats suggèrent que la prise de poids induite par le B[a]P est peut-être liée à son action inhibitrice sur le LSR et le LDL-R, ainsi que sur l’ABCA1 et le métabolisme des lipoprotéines hépatiques, ce qui conduit à un statut lipidique modifié chez les souris traitées au B[a]P, donnant ainsi un nouvel éclairage sur les mécanismes sous-jacents de la contribution des polluants tels que le B[a]P à la perturbation de l'homéostasie lipidique, susceptible de contribuer à la dyslipidémie associée à l'obésité / Obesity is a multifactorial disorder that represents a significant risk factor for many pathologies including cardiovascular diseases, diabetes and neurodegenerative diseases. Recent epidemiological studies suggest potential obesogenic effects of environmental contaminants, but little information is available on their potential effect on hepatic lipoprotein metabolism. The objective of this study was to determine the effect of the common environmental pollutants, belonging to polycyclic aromatic hydrocarbon (HAP) on three lipoprotein receptors, the LDL-receptor (LDL-R), the scavenger receptor B1 (SRB1) and the lipolysis-stimulated lipoprotein receptor (LSR) as well as the ATP binding cassette transporters A1 (ABCA1) and G1 (ABCG1) using cell and/or animal models. Immunoblot and immunofluorescence in vitro studies revealed that exposure of Hepa1-6 to benzo[a]pyrene (B[a]P) significantly decreased LSR, LDL-R and ABCA1 protein levels, whereas no significant changes in protein levels and LSR activity where observed upon cell treatment with pyrene or phenanthrene. Real-time PCR analysis, lactacystin and chloroquine studies revealed that this effect was due primarily to increased proteasome-mediated degradation rather than to decreased transcription or to increased lysosomal degradation. Furthermore, ligand blots revealed that lipoproteins exposed to B[a]P displayed markedly decreased binding to LSR or LDL-R. C57Bl/6RJ mice were treated with B[a]P (0.5 mg/kg, i.p) every 48 h for 15 days. The increased weight gain observed was accompanied by increased plasma triglycerides and cholesterol levels, increased liver cholesterol content, and decreased LDL-R, ABCA1, ABCG1 and SR-B1 protein levels in B[a]P-treated animals as compared to controls. Correlations observed between hepatic LSR and LDL-R levels in control mice were no longer observed in B[a]P treated mice, suggesting a potential dysregulation of hepatic lipoprotein metabolism.Taken together, these results suggest that B[a]P-induced weight gain may be due its inhibitory action on LSR and LDL-R, as well as ABCA1 and lipoprotein metabolism in the liver, which leads to the modified lipid status in B[a]P-treated mice, thus providing new insight into mechanisms underlying the involvement of pollutants such as B[a]P in the disruption of lipid homeostasis, potentially contributing to dyslipidemia associated with obesity

Un prélèvement efficace de K+ à partir du sol est essentiel au développement des végétaux. Sur un sol riche en NaCl, le maintien d'un prélèvement sélectif et efficace de K+ à partir du sol et le contrôle de l'exportation de Na+ par la racine vers les feuilles constituent des fonctions essentielles pour la survie de la plante. Chez les plantes, les transporteurs HKT (High-affinity K+ Transporters) sont classés en deux sous-familles sur des bases phylogénétiques et de sélectivité ionique. Les membres de la sous-famille 1 transportent sélectivement Na+. Plusieurs d'entre eux ont été identifiés comme des acteurs majeurs de l'adaptation des plantes aux fortes salinités du sol en prévenant l'accumulation de Na+ dans les parties aériennes. Les membres de la sous-famille 2 co-transportent Na+ et K+. Leur rôle dans la plante, notamment dans le transport de K+, est encore mal compris. Je me suis intéressé à différents systèmes de transports de K+ et Na+, appartenant essentiellement à la famille HKT chez le riz. La caractérisation que j'ai effectuée a fait appel à plusieurs approches : électrophysiologie (voltage-clamp après expression en ovocyte de xénope), biologie cellulaire, génétique inverse et PCR en temps réel. L'analyse de l'expression par RT-PCR en temps réel de toute la famille HKT (4 membres dans chacune des deux sous-familles) a montré que ces transporteurs sont différemment exprimés au niveau des racines et des feuilles, et que leur niveau de transcrits est fortement et differentiellement régulé en conditions de stress salin ou osmotique et en présence d'hormones, ce qui suggère que ces différents systèmes jouent des rôles propres et diversifiés dans la plante. L'analyse plus détaillée d'OsHKT2;4, a montré par expression hétérologue dans l'ovocyte de xénope que ce système possède des propriétés fonctionnelles originales: il transporte sélectivement K+ à faibles concentrations de Na+, mais co-transporte Na+ et K+ à fortes concentrations de Na+ (>10 mM). L'analyse de l'expression d'OsHKT2;4 a révélé que ce transporteur est surexprimé en condition de carence en K+ et de stress salin, suggérant qu'OsHKT2;4 pourrait jouer un rôle important dans le transport de K+ dans ces deux conditions. Enfin, un patron d'expression nouveau pour un transporteur HKT a été révélé par l'analyse de plantes transgéniques exprimant le promoteur d'OsHKT2;4 fusionné aux gènes rapporteurs GUS ou GFP : en plus d'une localisation classique dans les tissus conducteurs, une forte expression est observée dans les stomates des gaines et des limbes foliaires, suggérant un rôle dans l'osmocontractilité de ces cellules.Mots clés: Oryza sativa, transport de potassium, transporteur HKT, Na+-K+ co-transporteur, électrophysiologie, ovocyte de xénope, localisation tissulaire, PCR quantitative, stress salin / Efficient uptake of K+ from the soil solution is essential for plant development. When plants are grown on a soil rich in NaCl, the maintenance of an efficient and selective uptake of K+ and the control of Na+ export from roots to shoots are crucial for plant survival. In plants, transporters belonging to the HKT (Highaffinity K+ Transport) family have been sorted in two subfamilies based on phylogenetic grounds and functional properties. Subfamily 1 members transport selectively Na+. Several of them have been shown to play major roles in plant adaptation to salt stress by preventing excessive accumulation of Na+ in shoots. Subfamily 2 members are thought to co-transport Na+ and K+, at least when expressed in heterologous systems. Their roles in planta, especially their potential role in K+ transport, are still largely unknown. I have been interested in different K+ and/or Na+ transport systems in rice, mostly belonging to the HKT family. For their characterization, different approaches have been used: electrophysiology (two-electrode voltage-clamp after expression in Xenopus oocytes), cell biology, reverse genetics and real-time PCR. Realtime RT-PCR analyses on the whole family of rice HKT transporters (4 members in both subfamilies) showed that the expression level in roots and leaves of these different systems is variable, and is differentially regulated by salt and osmotic stresses as well as by hormonal treatments, which suggests that these transporters have diverse and differentiated functions in the plant. A detailed analysis of OsHKT2;4 revealed original functional properties: this HKT transporter was indeed shown to be K+-selectively in the presence of low external Na+, but to switch to Na+ and K+ co-transport mode at high (>10 mM) Na+ concentrations. Expression analysis of OsHKT2;4 showed that this transporter is overexpressed upon salt stress and K+ shortage, which suggests that it could play an important role in K+ transport in these two conditions. At last, a new expression pattern for an HKT transporter was evidenced through the analysis of transgenic rice plants expressing OsHKT2;4 promoter fused to the GUS or GFP reporter genes: in addition to a classical localization in vascular tissues, expression of OsHKT2;4 was observed in stomata, suggesting a role for OsHKT2;4 in osmotic regulation in these cells

Etliche Membran Transport Proteine (MTP) sind essentiell in den Plasmodium Blutstadien, und geraten zunehmend in den Fokus der Wirkstoffentwicklung. Die physiologischen Rollen der Transporter sind jedoch oft ungeklärt. In dieser Arbeit wurden mittels experimenteller Genetik funktionelle Charakteristika der MTPs untersucht. Am Maus Parasiten Plasmodium berghei und der Plasmodium falciparum Blutstadien-Kultur wurden vier MTPs ausgewählt: ein konservierter Folat Transporter (FT2), sowie eine P. falciparum-spezifisches P-Typ ATPase und zwei essentielle MTPs (CRT und ATP4). Diese Auswahl verkörpert ein breites Spektrum an MTP Kandidaten und reflektieren zudem das Potenzial und die Grenzen funktioneller Analysen von Plasmodium MTPs mittels reverser Genetik. Für den Folat Transporter 2 (FT2) wurde eine Kombination von transgenen Strategien auf P. berghei angewandt. Durch ein endogenes tag von FT2 wurde die Lokalisierung im Apicoplast, sowie dessen Expression über fast den kompletten Zyklus hinweg gezeigt. Nach der Deletion von FT2, wiesen die Parasiten einen Defekt während der Sporulation auf. Demzufolge bilden sich nur nicht infektiöse Sporozoiten, was letztendlich zur Unterbrechung des Lebenszyklus der Parasiten führt. Eine Aminophospholipid P-Typ ATPase, wurde mittels CRISPR/Cas9 in P. falciparum genetisch deletiert und die Mutante analysiert. Im Gegensatz zu den meisten vitalen P-Typ ATPasen erweist sich das Gen in den asexuellen Blutstadien als entbehrlich. Des Weiteren bilden die MTPs ATP4 und CRT einen einflussreichen Faktor bei Malaria-Therapien. Eine umfassende Analyse von räumlichen und zeitlichen Expressionsmustern von transgenen Parasiten mit mCherry-getaggten Proteinen zeigt ein Expression der beiden MTPs über die Blutstadien hinaus, was auf zusätzliche Funktionen in den jeweiligen Stadien verweist. Diese Studie trägt, basierend auf Lokalisation, Expression und funktioneller Deletion, zur funktionellen Entschlüsselung der vier untersuchten MTPs bei. / Many membrane transport proteins (MTP) are essential for Plasmodium infection and gain importance as candidate drug targets in malaria therapy, whereas the physiological functions often remain enigmatic. In this thesis, we applied experimental genetics to determine key characteristics of four Plasmodium MTPs. We employed the murine malaria model parasite Plasmodium berghei and in vitro blood cultures of Plasmodium falciparum. We selected one conserved MTP called FT2, which was previously shown to transport folate, a P-type ATPase that is specific for P. falciparum as well as two essential MTPs, CRT and ATP4. These targets exemplify the range of druggable candidates and illustrate the potential and limitations of reverse genetics to decipher their physiological roles. A combination of transgenic and knockout strategies was applied to the P. berghei folate transporter 2 (FT2). We show that endogenously tagged FT2 localises to the apicoplast membranes, and is broadly expressed throughout the parasite’s life cycle. Analysis of FT2-deficient parasites revealed a severe sporulation defect in the vector; the vast majority of ft2– oocysts form large intracellular vesicles which displace the cytoplasm. Very few sporozoites are generated and these are non-infectious to the mammalian host, resulting in a complete arrest of Plasmodium transmission. A candidate aminophospholipid P-type ATPase, was assessed by a CRISPR/Cas9-mediated gene disruption. Compared to many vital P-type ATPases this gene is dispensable for asexual blood replication. Two MTPs, ATP4 and CRT are prime targets for antimalarial therapies. A comprehensive spatio-temporal expression analysis of transgenic parasites expressing mCherry-tagged proteins revealed expression beyond blood infection, indicative of functions in additional parasite stages. The findings of this study contribute towards a better understanding of the roles of the four MTPs based on localisation, expression and functional deletion.