Interactions between the NO-Citrulline Cycle and Brain-derived Neurotrophic Factor in Differentiation of Neural Stem Cells

The diffusible messenger NO plays multiple roles in neuroprotection, neurodegeneration, and brain plasticity. Argininosuccinate synthase (AS) is a ubiquitous enzyme in mammals and the key enzyme of the NO-citrulline cycle, because it provides the substrate L-arginine for subsequent NO synthesis by inducible, endothelial, and neuronal NO synthase (NOS). Here, we provide evidence for the participation of AS and of the NO-citrulline cycle in the progress of differentiation of neural stem cells (NSC) into neurons, astrocytes, and oligodendrocytes. AS expression and activity and neuronal NOS expression, as well as L-arginine and NOx production, increased along neural differentiation, whereas endothelial NOS expression was augmented in conditions of chronic NOS inhibition during differentiation, indicating that this NOS isoform is amenable to modulation by extracellular cues. AS and NOS inhibition caused a delay in the progress of neural differentiation, as suggested by the decreased percentage of terminally differentiated cells. On the other hand, BDNF reversed the delay of neural differentiation of NSC caused by inhibition of NOx production. Alikely cause is the lack of NO, which up-regulated p75 neurotrophin receptor expression, a receptor required for BDNF-induced differentiation of NSC. We conclude that the NO-citrulline cycle acts together with BDNF for maintaining the progress of neural differentiation.

NF-κB Drives the Synthesis of Melatonin in RAW 264.7 Macrophages by Inducing the Transcription of the Arylalkylamine-N-Acetyltransferase (AA-NAT) Gene

We demonstrate that during inflammatory responses the nuclear factor kappa B (NF-kappa B) induces the synthesis of melatonin by macrophages and that macrophage-synthesized melatonin modulates the function of these professional phagocytes in an autocrine manner. Expression of a DsRed2 fluorescent reporter driven by regions of the aa-nat promoter, that encodes the key enzyme involved in melatonin synthesis (arylalkylamine-N-acetyltransferase), containing one or two upstream kappa B binding sites in RAW 264.7 macrophage cell lines was repressed when NF-kappa B activity was inhibited by blocking its nuclear translocation or its DNA binding activity or by silencing the transcription of the RelA or c-Rel NF-kappa B subunits. Therefore, transcription of aa-nat driven by NF-kappa B dimers containing RelA or c-Rel subunits mediates pathogen-associated molecular patterns (PAMPs) or pro-inflammatory cytokine-induced melatonin synthesis in macrophages. Furthermore, melatonin acts in an autocrine manner to potentiate macrophage phagocytic activity, whereas luzindole, a competitive antagonist of melatonin receptors, decreases macrophage phagocytic activity. The opposing functions of NF-kappa B in the modulation of AA-NAT expression in pinealocytes and macrophages may represent the key mechanism for the switch in the source of melatonin from the pineal gland to immune-competent cells during the development of an inflammatory response.

Ectopic expression of a fruit phytoene synthase from Citrus paradisi Macf. promotes abiotic stress tolerance in transgenic tobacco

Abscisic acid (ABA) is an important regulator of plant responses to environmental stresses and an absolute requirement for stress tolerance. Recently, a third phytoene synthase (PSY3) gene paralog was identified in monocots and demonstrated to play a specialized role in stress-induced ABA formation, thus suggesting that the first committed step in carotenogenesis is a key limiting step in ABA biosynthesis. To examine whether the ectopic expression of PSY, other than PSY3, would similarly affect ABA level and stress tolerance, we have produced transgenic tobacco containing a fruit-specific PSY (CpPSY) of grapefruit (Citrus paradisi Macf.). The transgenic plants contained a single- or double-locus insertion and expressed CpPSY at varying transcript levels. In comparison with the wild-type plants, the CpPSY expressing transgenic plants showed a significant increase on root length and shoot biomass under PEG-, NaCl- and mannitol-induced osmotic stress. The enhanced stress tolerance of transgenic plants was correlated with the increased endogenous ABA level and expression of stress-responsive genes, which in turn was correlated with the CpPSY copy number and expression level in different transgenic lines. Collectively, these results provide further evidence that PSY is a key enzyme regulating ABA biosynthesis and that the altered expression of other PSYs in transgenic plants may provide a similar function to that of the monocot's PSY3 in ABA biosynthesis and stress tolerance. The results also pave the way for further use of CpPSY, as well as other PSYs, as potential candidate genes for engineering tolerance to drought and salt stress in crop plants.

Role of xanthophyll and water-water cycles in the protection of photosynthetic apparatus in Arbutus unedo and Arabidopsis thaliana

Photosynthetic organisms have sought out the delicate balance between efficient light harvesting under limited irradiance and regulated energy dissipation under excess irradiance. One of the protective mechanisms is the thermal energy dissipation through the xanthophyll cycle that may transform harmlessly the excitation energy into heat and thereby prevent the formation of damaging active oxygen species (AOS). Violaxanthin deepoxidase (VDE) converts violaxanthin (V) to antheraxanthin (A) and zeaxanthin (Z) defending the photosynthetic apparatus from excess of light. Another important biological pathway is the chloroplast water-water cycle, which is referred to the electrons from water generated in PSII reducing atmospheric O2 to water in PSI. This mechanism is active in the scavenging of AOS, when electron transport is slowed down by the over-reduction of NADPH pool. The control of the VDE gene and the variations of a set of physiological parameters, such as chlorophyll florescence and AOS content, have been investigated in response to excess of light and drought condition using Arabidopsis thaliana and Arbutus unedo.. Pigment analysis showed an unambiguous relationship between xanthophyll de-epoxidation state ((A+Z)/(V+A+Z)) and VDE mRNA amount in not-irrigated plants. Unexpectedly, gene expression is higher during the night when xanthophylls are mostly epoxidated and VDE activity is supposed to be very low than during the day. The importance of the water-water cycle in protecting the chloroplasts from light stress has been examined through Arabidopsis plant with a suppressed expression of the key enzyme of the cycle: the thylakoid-attached copper/zinc superoxide dismutase. The analysis revealed changes in transcript expression during leaf development consistent with a signalling role of AOS in plant defence responses but no difference was found any in photosynthesis efficiency or in AOS concentration after short-term exposure to excess of light. Environmental stresses such as drought may render previously optimal light levels excessive. In these circumstances the intrinsic regulations of photosynthetic electron transport like xanthophyll and water-water cycles might modify metabolism and gene expression in order to deal with increasing AOS.

Planktonic potential CO2 emission calculation: preliminary results by applying an adapted enzymatic methodology to marine ecosystems

[EN]Isocitrate Dehydrogenase (IDH) is a key enzyme in the Krebs cycle, being responsible for the production of one of the three CO2 molecules related to cellular respiration. In order to measure the potential CO2 production linked to the marine planktonic community we have adapted an enzymatic methodology. Preliminary results show that different proportions of autotrophs, heterotrophs and mixotrophs and their metabolic pathways, lead to different relationships between potential CO2 emission and potential O2 consumption during cellular respiration. Although more experiments need to be made, this methodology is leading to a better understanding of cellular respiration in marine samples and their impact on the food chain, vertical Carbon flux and the current sequestering capacity for anthropogenic CO2.

Influence of climate changes on cross allergies: possible involvement of pollen transglutaminase

Allergies are a complex of symptoms derived from altered IgE-mediated reactions of the immune system towards substances known as allergens. Allergic sensibilization can be of food or respiratory origin and, in particular, apple and hazelnut allergens have been identified in pollens or fruits. Allergic cross-reactivity can occur in a patient reacting to similar allergens from different origins, justifying the research in both systems as in Europe a greater number of people suffers from apple fruit allergy, but little evidence exists about pollen. Apple fruit allergies are due to four different classes of allergens (Mal d 1, 2, 3, 4), whose allergenicity is related both to genotype and tissue specificity; therefore I have investigated their presence also in pollen at different time of germination to clarify the apple pollen allergenic potential. I have observed that the same four classes of allergens found in fruit are expressed at different levels also in pollen, and their presence might support that the apple pollen can be considered allergenic as the fruit, deducing that apple allergy could also be indirectly caused by sensitization to pollen. Climate changes resulting from increases in temperature and air pollution influence pollen allergenicity, responsible for the dramatic raise in respiratory allergies (hay fever, bronchial asthma, conjunctivitis). Although the link between climate change and pollen allergenicity is proven, the underlying mechanism is little understood. Transglutaminases (TGases), a class of enzymes able to post-translationally modify proteins, are activated under stress and involved in some inflammatory responses, enhancing the activity of pro-inflammatory phospholipase A2, suggesting a role in allergies. Recently, a calcium-dependent TGase activity has been identified in the pollen cell wall, raising the possibility that pollen TGase may have a role in the modification of pollen allergens reported above, thus stabilizing them against proteases. This enzyme can be involved also in the transamidation of proteins present in the human mucosa interacting with surface pollen or, finally, the enzyme itself can represent an allergen, as suggested by studies on celiac desease. I have hypothesized that this pollen enzyme can be affected by climate changes and be involved in exhacerbating allergy response. The data presented in this thesis represent a scientific basis for future development of studies devoted to verify the hypothesis set out here. First, I have demonstrated the presence of an extracellular TGase on the surface of the grain observed either at the apical or the proximal parts of the pollen-tube by laser confocal microscopy (Iorio et al., 2008), that plays an essential role in apple pollen-tube growth, as suggested by the arrest of tube elongation by TGase inhibitors, such as EGTA or R281. Its involvement in pollen tube growth is mainly confirmed by the data of activity and gene expression, because TGase showed a peak between 15 min and 30 min of germination, when this process is well established, and an optimal pH around 6.5, which is close to that recorded for the germination medium. Moreover, data show that pollen TGase can be a glycoprotein as the glycosylation profile is linked both with the activation of the enzyme and with its localization at the pollen cell wall during germination, because from the data presented seems that the active form of TGase involved in pollen tube growth and pollen-stylar interaction is more exposed and more weakly bound to the cell wall. Interestingly, TGase interacts with fibronectin (FN), a putative SAMs or psECM component, inducing possibly intracellular signal transduction during the interaction between pollen-stylar occuring in the germination process, since a protein immunorecognised by anti-FN antibody is also present in pollen, in particular at the level of pollen grain cell wall in a punctuate pattern, but also along the shank of the pollen tube wall, in a similar pattern that recalls the signal obtained with the antibody anti TGase. FN represents a good substrate for the enzyme activity, better than DMC usually used as standard substrate for animal TGase. Thus, this pollen enzyme, necessary for its germination, is exposed on the pollen surface and consequently can easily interact with mucosal proteins, as it has been found germinated pollen in studies conducted on human mucus (Forlani, personal communication). I have obtained data that TGase activity increases in a very remarkable way when pollen is exposed to stressful conditions, such as climate changes and environmental pollution. I have used two different species of pollen, an aero allergenic (hazelnut, Corylus avellana) pollen, whose allergenicity is well documented, and an enthomophylus (apple, Malus domestica) pollen, which is not yet well characterized, to compare data on their mechanism of action in response to stressors. The two pollens have been exposed to climate changes (different temperatures, relative humidity (rH), acid rain at pH 5.6 and copper pollution (3.10 µg/l)) and showed an increase in pollen surface TGase activity that is not accompanied to an induced expression of TGase immunoreactive protein with AtPNG1p. Probably, climate change induce an alteration or damage to pollen cell wall that carries the pollen grains to release their content in the medium including TGase enzyme, that can be free to carry out its function as confirmed by the immunolocalisation and by the in situ TGase activity assay data; morphological examination indicated pollen damage, viability significantly reduced and in acid rain conditions an early germination of apple pollen, thus possibly enhancing the TGase exposure on pollen surface. Several pollen proteins were post-translationally modified, as well as mammalian sPLA2 especially with Corylus pollen, which results in its activation, potentially altering pollen allergenicity and inflammation. Pollen TGase activity mimicked the behaviour of gpl TGase and AtPNG1p in the stimulation of sPLA2, even if the regulatory mechanism seems different to gpl TGase, because pollen TGase favours an intermolecular cross-linking between various molecules of sPLA2, giving rise to high-molecular protein networks normally more stable. In general, pollens exhibited a significant endogenous phospholipase activity and it has been observed differences according to the allergenic (Corylus) or not-well characterized allergenic (Malus) attitude of the pollen. However, even if with a different intensity level in activation, pollen enzyme share the ability to activate the sPLA2, thus suggesting an important regulatory role for the activation of a key enzyme of the inflammatory response, among which my interest was addressed to pollen allergy. In conclusion, from all the data presented, mainly presence of allergens, presence of an extracellular TGase, increasing in its activity following exposure to environmental pollution and PLA2 activation, I can conclude that also Malus pollen can behave as potentially allergenic. The mechanisms described here that could affect the allergenicity of pollen, maybe could be the same occurring in fruit, paving the way for future studies in the identification of hyper- and hypo- allergenic cultivars, in preventing environmental stressor effects and, possibly, in the production of transgenic plants.

Cross-Talk tra Bifidobacterium e intestino umano: impatto sulle attività health promoting

Bifidobacteria constitute up to 3% of the total microbiota and represent one of the most important healthpromoting bacterial groups of the human intestinal microflora. The presence of Bifidobacterium in the human gastrointestinal tract has been directly related to several health-promoting activities; however, to date, no information about the specific mechanisms of interaction with the host is available. The first health-promoting activities studied in these job was the oxalate-degrading activity. Oxalic acid occurs extensively in nature and plays diverse roles, especially in pathological processes. Due to its highly oxidizing effects, hyper absorption or abnormal synthesis of oxalate can cause serious acute disorders in mammals and be lethal in extreme cases. Intestinal oxalate-degrading bacteria could therefore be pivotal in maintaining oxalate homeostasis, reducing the risk of kidney stone development. In this study, the oxalate-degrading activity of 14 bifidobacterial strains was measured by a capillary electrophoresis technique. The oxc gene, encoding oxalyl-CoA decarboxylase, a key enzyme in oxalate catabolism, was isolated by probing a genomic library of B. animalis subsp. lactis BI07, which was one of the most active strains in the preliminary screening. The genetic and transcriptional organization of oxc flanking regions was determined, unravelling the presence of other two independently transcribed open reading frames, potentially responsible for B. animalis subsp. lactis ability to degrade oxalate. Transcriptional analysis, using real-time quantitative reverse transcription PCR, revealed that these genes were highly induced in cells first adapted to subinhibitory concentrations of oxalate and then exposed to pH 4.5. Acidic conditions were also a prerequisite for a significant oxalate degradation rate, which dramatically increased in oxalate pre-adapted cells, as demonstrated in fermentation experiments with different pH-controlled batch cultures. These findings provide new insights in the characterization of oxalate-degrading probiotic bacteria and may support the use of B. animalis subsp. lactis as a promising adjunct for the prophylaxis and management of oxalate-related kidney disease. In order to provide some insight into the molecular mechanisms involved in the interaction with the host, in the second part of the job, we investigated whether Bifidobacterium was able to capture human plasminogen on the cell surface. The binding of human plasminogen to Bifidobacterium was dependent on lysine residues of surface protein receptors. By using a proteomic approach, we identified six putative plasminogen-binding proteins in the cell wall fraction of three strain of Bifidobacterium. The data suggest that plasminogen binding to Bifidobactrium is due to the concerted action of a number of proteins located on the bacterial cell surface, some of which are highly conserved cytoplasmic proteins which have other essential cellular functions. Our findings represent a step forward in understanding the mechanisms involved in the Bifidobacterium-host interaction. In these job w studied a new approach based on to MALDI-TOF MS to measure the interaction between entire bacterial cells and host molecular target. MALDI-TOF (Matrix Assisted Laser Desorption Ionization-Time of Flight)—mass spectrometry has been applied, for the first time, in the investigation of whole Bifidobacterium cells-host target proteins interaction. In particular, by means of this technique, a dose dependent human plasminogen-binding activity has been shown for Bifidobacterium. The involvement of lysine binding sites on the bacterial cell surface has been proved. The obtained result was found to be consistent with that from well-established standard methodologies, thus the proposed MALDI-TOF approach has the potential to enter as a fast alternative method in the field of biorecognition studies involving in bacterial cells and proteins of human origin.

Valutazione dell'impatto della farmacogenetica in ambito di Sanità Pubblica. Analisi del polimorfismo genetico dell'enzima CYP2D6 metabolizzante xenobiotici.

Pharmacogenetic testing provides an outstanding opportunity to improve prescribing safety and efficacy. In Public health policy pharmacogenetics is relevant for personalized therapy and to maximize therapeutic benefit minimizing adverse events. CYP2D6 is known to be a key enzyme responsible for the biotransformation of about 25-30% of extensively used drugs and genetic variations in genes coding for drug-metabolizing enzymes might lead to adverse drug reactions, toxicity or therapeutic failure of pharmacotherapy. Significant interethnic differences in CYP2D6 allele distribution are well established, but immigration is reshaping the genetic background due to interethnic admixture which introduces variations in individual ancestry resulting in distinct level of population structure. The present thesis deals with the genetic determination of the CYP2D6 alleles actually present in the Emilia-Romagna resident population providing insights into the admixture process. A random sample of 122 natives and 175 immigrants from Africa, Asia and South America where characterized considering the present scenario of migration and back migration events. The results are consistent with the known interethnic genetic variation, but introduction of ethnic specific variants by immigrants predicts a heterogeneous admixed population scenario requiring, for drugs prescription and pharmacogenetics studies, an interdisciplinary approach applied in a properly biogeographical and anthropological frame. To translate pharmacogenetics knowledge into clinical practice requires appropriated public health policies, possibly guiding clinicians to evaluate prospectively which patients have the greatest probability of expressing a variant genotype.

Untersuchungen zur rekombinanten Expression und Faltung pflanzlicher Tyrosinasen

In ihrer dualen Funktion als Monophenolhydroxylase (EC 1.14.18.1) und Diphenoloxidase (EC 1.10.3.1) ist die Tyrosinase das Schlüsselenzym der Melanogenese, der Synthese des Melanins, und übernimmt damit quer durch alle Organismenreiche Aufgaben von der Pigmentierung bis hin zu einer Beteiligung an der Immunantwort. Sie zählt, zusammen mit den Catecholoxidasen und Hämocyaninen, zu den Typ-3-Kupfer-Proteinen, die sich durch ein Aktives Zentrum auszeichnen, das in der Lage ist, Sauerstoff und phenolische Substrate reversibel zwischen zwei Kupfer-Ionen zu binden. Bisher konnte weder die Funktion der pflanzlichen Tyrosinase genau identifiziert, noch die Struktur eines solchen Enzyms aufgeklärt werden. Mit dem späteren Ziel, durch eine röntgenkristallographische Analyse die zugrunde liegende strukturelle Ursache der zusätzlichen Monophenolhydroxylase-Aktivität von Tyrosinasen gegenüber reinen Catecholoxidasen ermitteln zu können, wurde in dieser Arbeit ein bakterielles Expressionssystem entwickelt, das zur Herstellung einer rekombinanten Tyrosinase oder Polyphenoloxidase (PPO) aus Spinacia oleracea (Spinat) für die Kristallisation verwendet werden kann. Das rekombinante Protein wurde in Form von Inclusion Bodies isoliert, anhand einer Affinitätschromatographie aufgereinigt und in anschließende Rückfaltungsexperimente eingesetzt. In einer parallelen Versuchsreihe konnte Spinat, aufgrund seiner hohen Tyrosinaseaktivität, als geeignetes Objekt für die Isolation des nativen Enzyms identifiziert werden. Im Anschluss an eine Thylakoidpräparation, Solubilisierung der Thylakoidmembranen und Fällung des Proteins mit Ammoniumsulfat, wurden Experimente zur weiteren Anreicherung der Tyrosinase-Aktivität über eine Anionenaustausch-Chromatographie und zur Etablierung einiger nachfolgender Aufreinigungsschritte durchgeführt.

Nachweis und Charakterisierung der Ketocarotinoidakkumulation in Zygosporen des Modellorganismus Chlamydomonas reinhardtii

Ketocarotinoide sind in den Dauerstadien vieler Grünalgen anzutreffen und aufgrund ihres hohen antioxidativen Potentials vermutlich von großer Bedeutung für deren Überleben unter ungünstigen Umweltbedingungen. Daneben ist die Aufnahme von Ketocarotinoiden im Zuge der Nahrungskette für verschiedene Tiere lebensnotwendig. Trotz zahlreicher Untersuchungen des Biosynthesewegs der Ketocarotinoide, vorwiegend in der Grünalge Haematococcus pluvialis, sind viele grundlegende Aspekte der Synthese nicht verstanden. Dazu zählt neben dem genauen Reaktionsmechanismus des ketolierenden Enzyms ß-Carotin-Ketolase (BKT) vor allem der noch nicht aufgeklärte Zusammenhang zwischen Lipidsynthese und Ketocarotinoidakkumulation. Nach der Entdeckung eines zur BKT aus H. pluvialis homologen Gens in einer EST-Datenbank des Modellorganismus Chlamydomonas reinhardtii wurden im Rahmen der vorliegenden Forschungsarbeit die als orange-rot beschrieben Zygosporen von C. reinhardtii als mögliches ketocarotinoidhaltiges Zellstadium untersucht. Dabei wurden für C. reinhardtii erstmals Ketocarotinoide in Konzentrationen bis zu einem Femtomol pro Zelle nachgewiesen und mittels HPLC-Analytik, chemischer Derivatisierung und Massenspektrometrie zweifelsfrei identifiziert. Es wurden, in aufsteigender Quantität, drei Ketocarotinoide detektiert: Canthaxanthin, Astaxanthin und 4-Ketolutein. Letzteres wurde bisher selten in anderen ketocarotinoidakkumulierenden Organismen beschrieben und stellt, im Gegensatz zu den vom ß-Carotin abgeleiteten Pigmenten Astaxanthin und Canthaxanthin, ein Pigment des α-Carotin-Zweiges dar. Astaxanthin und 4-Ketolutein wurden vor allem in Form von Pigment-Fettsäureestern nachgewiesen. Mit Hilfe von Paarungsansätzen mit der lor1-Mutante, die keine α-Carotinoide synthetisieren kann, und Vergleichen mit Ketocarotinoiden aus H. pluvialis konnte gezeigt werden, dass 4 Ketolutein nur als Monoacylester in der Alge vorliegt, während Astaxanthin sowohl als Monoacyl- wie auch als Diacylester anzutreffen ist. Ketocarotinoide wurden innerhalb der ersten 14 Tage der Zygotenreife gebildet. Transmissionselektronenmikroskopische Aufnahmen der Zygoten dokumentierten, dass damit ein starker Umbau der Zelle einherging, der sich vor allem in der Reduktion des Chloroplasten und der Bildung von Lipidtröpfchen darstellte. Letztere nahmen bei reifen Zygosporen den größten Teil des Zelllumens ein und wurden mittels dünnschichtchromatografischer Analysen als Neutralfette identifiziert. Der sinkende Zellgehalt an Carotinoiden im Zuge der Zygosporenreifung und Inhibitorexperimente an reifenden Zygoten mittels Norflurazon zeigten, dass für die Ketocarotinoidakkumulation keine Neusynthese von Carotinoiden nötig ist und lassen die Hypothese zu, dass C. reinhardtii die im Zuge der Chloroplastenreduktion freigesetzten Photosynthese-Carotinoide als Substrate für die Ketocarotinoidsynthese verwendet. Physiologische Bedeutung könnte den Ketocarotinoiden vor allem beim Schutz der Speicherlipide vor Peroxidation durch reaktive Sauerstoffspezies zukommen. Diese Reservestoffe stellen die Energieversorgung während des Auskeimens der Zellen sicher. Durch den im Rahmen der vorliegenden Forschungsarbeit dokumentierten Nachweis der Ketocarotinoidakkumulation in C. reinhardtii können die Ketocarotinoidsynthese und vor allem der Zusammenhang von Lipid- und Ketocarotinoidakkumulation zukünftig mit Hilfe der für diesen Modellorganismus vorliegenden umfangreichen molekulargenetischen Methoden detailliert untersucht werden.

Identifizierung und Quantifizierung der Ketocarotinoide in Dauerstadien von Grünalgen und Ketocarotinoidbiosynthese im Modellorganismus Chlamydomonas reinhardtii

Grünalgen bilden zur Überdauerung schlechter Umweltbedingungen Ruhestadien, die sich durch Ausbildung einer festen Zellwand, die Reduktion des Plastiden und die starke Akkumulation von Speicherfetten und Ketocarotinoiden im Zytosol auszeichnen. Obwohl Ketocarotinoide in Grünalgen seit über vierzig Jahren beforscht werden, gab es hierzu noch wenige molekularbiologische Untersuchungen. Im Vorfeld meiner Promotion wurde durch unsere Arbeitsgruppe entdeckt, dass auch der molekular gut zugängliche Modellorganismus Chlamydomonas reinhardtii im Zygotenstadium große Mengen an Ketocarotinoiden bildet. Neben dem zu erwartenden Ketocarotinoid Astaxanthin fanden wir große Mengen des bisher nur in einer Grünalge beschriebenen 4-Ketoluteins. Vorversuche ließen die Vermutung aufkommen, dass dieses Pigment bei der Untersuchung der Pigmentausstattung in Dauerstadien von vielen Grünalgen bisher übersehen wurde. rnIn der vorliegenden Arbeit wurde daher zunächst die Pigmentzusammensetzung von Dauerstadien der bereits gut untersuchten Grünalgen Muriella zofingiensis und Scenedesmus rubescens durch Vergleich mit dem Ketocarotinoidmuster aus Dauerstadien von C. reinhardtii und Fritschiella tuberosa reevaluiert und dabei erstmals das Vorkommen signifikanter Mengen an 4-Ketolutein nachgewiesen. Außerdem zeigte sich, dass die als bisheriger Modellorganismus der Ketocarotinoidbiosynthese in Grünalgen sehr gut untersuchte Alge Haematococcus pluvialis eher eine Ausnahme darstellt, da ihre Dauerstadien als einzige der hier untersuchten Algen nur minimale Mengen von 4 Ketolutein aufwiesen. Diese Beobachtungen machen es sehr wahrscheinlich, dass die Fähigkeit zur Bildung von 4-Ketolutein unter den Grünalgen wesentlich weiter verbreitet ist als bisher angenommen. Das sekundäre Carotinoid 4-Ketolutein kam in den Dauerstadien der Grünalgen neben seiner freien Form ausschließlich als Monoacylester vor, im Gegensatz zu Astaxanthin, das als mono- und diacylierte Form auftrat. rnÜber die Analyse der Pigmentausstattung hinaus konnten die entscheidenden Schritte des Synthesewegs der Ketocarotinoide in C. reinhardtii durch funktionelle Charakterisierung der beteiligten Enzyme in Bakterien aufgeklärt werden. Als Basis für die Charakterisierungen wurde ein umfangreiches Portfolio von carotinogenen E. coli-Bakterien etabliert, darunter α Carotin und Lutein produzierende Stämme, die bisher nicht zur Verfügung standen. Das wurde durch die Klonierung der Lycopinzyklase (OluLCY) aus der Grünalge Ostreococcus lucimarinus möglich, die eine Sonderolle unter den Zyklasen einnimmt, da sie die Lycopin-β-Zyklase und Lycopin-ε-Zyklase in einem Fusionsenzym vereint. Vorteile dieses Fusionsenzyms sind die Expressionskontrolle durch nur einen Promotor und die weitgehend konstante Stöchiometrie seiner Produkte α-Carotin und β-Carotin, was die OluLCY für die biotechnologische Anwendung prädestiniert.rnDie funktionelle Charakterisierung der Carotinoidbiosyntheseenzyme aus C. reinhardtii umfasste das Schlüsselenzym der Ketocarotinoidbiosynthese, die β-Carotin-Ketolase (BKT), sowie die Carotinoid-Hydroxylasen CHYB, CYP97A5 und CYP97C3. Dabei wurde für das BKT-Enzym aus C. reinhardtii nachgewiesen, dass es nicht nur die Ketolierung von β Carotin zu Canthaxanthin und von Zeaxanthin zu Astaxanthin, sondern auch die Bildung der von α-Carotin abgeleiteten Ketocarotinoide wie 4-Keto-α-Carotin und 4 Ketolutein katalysieren kann.rn

Funktionelle Charakterisierung der Casein Kinase 2 in der Etablierung und Aufrechterhaltung peripherer Toleranz durch dendritische Zellen

Dendritische Zellen (DCs) nehmen eine Schlüsselrolle in unserem Immunsystem ein, indem DCs sowohl Immunität, als auch Toleranz induzieren können. Im Falle der Immunität sind DCs in der Lage die Differenzierung der verschiedenen T-Helferzellen, wie Th1-, Th2- und Th17-Zellen zu steuern und tragen so zu der Qualität einer Immunantwort bei. Auf der anderen Seite können DCs in Gegenwart von TGF-β, IDO und Retinsäure die Differenzierung von regulatorischen T-Zellen induzieren und tragen somit zur Aufrechterhaltung der peripheren Toleranz bei. Insbesondere in den Darm-assoziierten lymphatischen Geweben (GALT) müssen DCs unverhältnismäßige Immunantworten gegen harmlose Antigene aus der Nahrung und kommensale Bakterien verhindern, während gegen Pathogene schützende Immunantworten induziert werden müssen. Auf Grund dieser entgegengesetzten Funktionen der DCs wollten wir die molekularen Mechanismen der DCs untersuchen, die der Regulation von Immunität und Toleranz zu Grunde liegen. Insbesondere der Wnt-Signalweg ist für die Aufrechterhaltung der peripheren Toleranz im GALT von Bedeutung. Da die Casein Kinase 2 in diesem Signalweg entscheidend beteiligt ist, haben wir die CK2-Funktion konditionell, unter der Kontrolle des CD11c-Promotors, deletiert. Hierfür haben wir CD11c-cre Mäuse mit Mäusen verpaart, welche ein von loxP-Signalsequenzen flankiertes Ck2β Gen (CK2β-fl/fl) tragen. Die konditionelle Deletion der CK2-Funktion in DCs, führte zu einer verstärkten Expression der kostimulatorischen Moleküle (wie CD40, CD80, CD86) und der Zytokine IL-6 und IL-12 unter „steady-state“ Bedingungen. Detaillierte Untersuchungen der T-Zellen in CD11c-cre x CK2β-fl/fl Mäusen zeigte eine deutlich reduzierte naive T-Zellpopulation, einhergehend mit einer erhöhten Th1- und Th17-Differenzierung. Speziell in den mesenterialen Lymphknoten konnte eine höhere Frequenz von T-bet+ und Rorγt+ CD4+ T-Zellen gefunden werden, welche große Mengen der Zytokine IFN-γ und IL-17 nach ex vivo Stimulation produzierten. Weiterführende in vivo Versuche, hier wurde das Modell der oralen Toleranz gewählt, zeigten das eine CK2-Deletion in DCs die Induktion einer oralen Toleranz verhindert. Unsere Daten zeigen eindeutig, dass die CK2 entscheidend in der Regulation der DC Homöostase und der Aufrechterhaltung der peripheren Toleranz beteiligt ist.

Mechanisms of resistance of tumor cells in response to treatment with the vacuolar H+-ATPase inhibitor archazolid B

Resistance of cancer cells towards chemotherapy is the major cause of therapy failure. Hence, the evaluation of cellular defense mechanisms is essential in the establishment of new chemotherapeutics. In this study, classical intrinsic and acquired as well as new resistance mechanisms relevant in the cellular response to the novel vacuolar H+-ATPase inhibitor archazolid B were investigated. Archazolid B, originally produced by the myxobacterium Archangium gephyra, displayed cytotoxicity in the low nanomolar range on a panel of cancer cell lines. The drug showed enhanced cytotoxic activity against nearly all cancerous cells compared to their non-cancerous pendants. With regards to ABC transporters, archazolid B was identified as a moderate substrate of ABCB1 (P-glycoprotein) and a weak substrate of ABCG2 (BCRP), whereas hypersensitivity was observed in ABCB5-expressing cells. The cytotoxic effect of archazolid B was shown to be independent of the cellular p53 status. However, cells expressing constitutively active EGFR displayed significantly increased resistance. Acquired drug resistance was studied by establishing an archazolid B-resistant MCF-7 cell line. Experiments showed that this secondary resistance was not conferred by aberrant expression or DNA mutations of the gene encoding vacuolar H+-ATPase subunit c, the direct target of archazolid B. Instead, a slight increase of ABCB1 and a significant overexpression of EGFR as well as reduced proliferation may contribute to acquired archazolid B resistance. For identification of new resistance strategies upon archazolid B treatment, omics data from bladder cancer and glioblastoma cells were analyzed, revealing drastic disturbances in cholesterol homeostasis, affecting cholesterol biosynthesis, uptake and transport. As shown by filipin staining, archazolid B led to accumulation of free cholesterol in lysosomes, which triggered sterol responses, mediated by SREBP-2 and LXR, including up-regulation of HMGCR, the key enzyme of cholesterol biosynthesis. Furthermore, inhibition of LDL uptake as well as impaired LDLR surface expression were observed, indicating newly synthesized cholesterol to be the main source of cholesterol in archazolid B-treated cells. This was proven by the fact that under archazolid B treatment, total free cholesterol levels as well as cell survival were significantly reduced by inhibiting HMGCR with fluvastatin. The combination of archazolid B with statins may therefore be an attractive strategy to circumvent cholesterol-mediated cell survival and in turn potentiate the promising anticancer effects of archazolid B.

A novel mode of action of the putative sphingosine kinase inhibitor 2-(p-hydroxyanilino)-4-(p-chlorophenyl) thiazole (SKI II): induction of lysosomal sphingosine kinase 1 degradation

Sphingosine kinase 1 (SK1) is a key enzyme in the generation of sphingosine 1-phosphate (S1P) which critically regulates a variety of important cell responses such as proliferation and migration. Therefore, inhibition of SK-1 has been suggested to be an attractive approach to treat tumor growth and metastasis formation.

Dihydropyrimidine dehydrogenase gene as a major predictor of severe 5-fluorouracil toxicity

The importance of polymorphisms in the dihydropyrimidine dehydrogenase (DPD) gene (DPYD) for the prediction of severe toxicity in 5-fluorouracil (5-FU) based chemotherapy has been controversially debated. As a key enzyme in the catabolism of 5-FU, DPD is the top candidate for pharmacogenetic studies on 5-FU toxicity, since a reduced DPD activity is thought to result in an increased half-life of the drug, and thus, an increased risk of toxicity. Here, we review the current knowledge on well-known and frequently studied DPYD variants such as the c.1905+1G>A splice site variant, as well as the recent discoveries of important functional variation in the noncoding regions of DPYD. We also outline future directions that are needed to further improve the risk assessment of 5-FU toxicity, in particular with respect to metabolic profiling and in the context of different combination therapeutic regimens, in which 5-FU is used today.