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Streptococcus pneumoniae (Pneumococcus) is one of the leading causes of childhood meningitis,pneumonia and sepsis. Despite the availability of childhood vaccination programs and antimicrobial agents, childhood pneumococcal meningitis is still a devastating illness with mortality rates among the highest of any cause of bacterial meningitis. Especially in low-income countries, where medical care is less accessible, mortality rates up to 50 % have been reported. In surviving patients, neurological sequelae, including hearing loss, focal neurological deficits and cognitive impairment, is reported in 30 to 50 %. Growing resistance of pneumococci towards conventional antibiotics emphasize the need for effective therapies and development of effective vaccines against Streptococcus pneumoniae. One major virulence factor of Streptococcus pneumoniae is the protein toxin Pneumolysin (PLY). PLY belongs to a family of structurally related toxins, the so-called cholesterol-dependent cytolysins (CDCs). Pneumolysin is produced by almost all clinical isolates of the bacterium. It is expressed during the late log phase of bacterial growth and gets released mainly through spontaneous autolysis of the bacterial cell. After binding to cholesterol in the host cell membranes, oligomerization of up to 50 toxin monomers and rearrangement of the protein structure, PLY forms large pores, leading to cell lysis in higher toxin concentrations. At sub-lytic concentrations, however, PLY mediates several other effects, such as activation of the classic complement pathway and the induction of apoptosis. First experiments with pneumococcal strains, deficient in pneumolysin, showed a reduced virulence of the organism, which emphasizes the contribution of this toxin to the course of bacterial meningitis and the urgent need for the understanding of the multiple mechanisms leading to invasive pneumococcal disease. The aim of this thesis was to shed light on the contribution of pneumolysin to the course of the disease as well as to the mental illness patients are suffering from after recovery from pneumococcal meningitis. Therefore, we firstly investigated the effects of sub-lytic pneumolysin concentrations onto primary mouse neurons, transfected with a GFP construct and imaged with the help of laser scanning confocal microscopy. We discovered two major morphological changes in the dendrites of primary mouse neurons: The formation of focal swellings along the dendrites (so-called varicosities) and the reduction of dendritic spines. To study these effects in a more complex system, closer to the in vivo situation, we established a reproducible method for acute brain slice culturing. With the help of this culturing method, we were able to discover the same morphological changes in dendrites upon challenge with sub-lytic concentrations of pneumolysin. We were able to reverse the seen alterations in dendritic structure with the help of two antagonists of the NMDA receptor, connecting the toxin´s mode of action to a non-physiological stimulation of this subtype of glutamate receptors. The loss of dendritic spines (representing the postsynapse) in our brain slice model could be verified with the help of brain slices from adult mice, suffering from pneumococcal meningitis. By immunohistochemical staining with an antibody against synapsin I, serving as a presynaptic marker, we were able to identify a reduction of synapsin I in the cortex of mice, infected with a pneumococcal strain which is capable of producing pneumolysin. The reduction of synapsin I was higher in these brain slices compared to mice infected with a pneumococcal strain which is not capable of producing pneumolysin, illustrating a clear role for the toxin in the reduction of dendritic spines. The fact that the seen effects weren´t abolished under calcium free conditions clarifies that not only the influx of calcium through the pneumolysin-pore is responsible for the alterations. These findings were further supported by calcium imaging experiments, where an inhibitor of the NMDA receptor was capable of delaying the time point, when the maximum of calcium influx upon PLY challenge was reached. Additionally, we were able to observe the dendritic beadings with the help of immunohistochemistry with an antibody against MAP2, a neuron-specific cytoskeletal protein. These observations also connect pneumolysin´s mode of action to excitotoxicity, as several studies mention the aggregation of MAP2 in dendritic beadings in response to excitotoxic stimuli. All in all, this is the first study connecting pneumolysin to excitotoxic events, which might be a novel chance to tie in other options of treatment for patients suffering from pneumococcal meningitis.
Mechanisms of apoptosis modulation and their contribution to genomic instability in tumor cells
(2004)
The concept of programmed cell death has been increasingly considered from various aspects since early 1970’s. Primarily, knowledge of apoptosis referred to morphological changes in which chromatin is condensed and increasingly fragmented, revealed as small structure in the nucleus. The membrane shrinks and the cell becomes dense as can be seen by flow cytometry. Interestingly, similar modes of cell deletion were observed in nematodes indicating that apoptosis is a highly conserved machinery. Three Caeonorhabditis elegans gene products are found to have high homology with mammalian apoptotic genes: CED-9 inhibits apoptosis and is related to bcl-2; CED-3 and CED-4 promote apoptosis and are related to caspase 9 and APAF-1. Apoptosis is not accidental death, but a highly controlled and medically important molecular process. More general terms such as ‘physiological’ or ‘regulated’ cell death cover different morphologies and sequences. Programmed suicide of cells that were subjected to toxic exogenous and endogenous stimuli plays a key role in understanding cancer development and its treatment. Apoptosis involves sequences of events that may overlap and play contradictory or antagonistic roles in cell death. Generally, the ability to trigger apoptotic processes in cancer cells would benefit an organism by keeping homeostasis intact. Programmed cell death is a regularly present mechanism, for instance, in lymphocyte recruitment in the thymus where immature lymphocytes may recognize host antigens. Therefore, such lymphocytes become apoptotic and are removed by macrophages. Removal prevents possible autoimmune diseases. Unlike apoptosis, necrosis is a passive process of cell death recognizable by membrane morphological changes and accompanied by leakage of intracellular material into intercellular space that may cause inflammation in the organism. Signals that may initiate apoptosis are generally classified into two groups: signals that launch extrinsic apoptotic pathways starting with aggregation of death receptors and intrinsic apoptotic pathways starting with disruption of intracellular homeostasis such as the release of mitochondrial factors or DNA degradation. Early in the process, apoptotic signals may lead to a broad range of signaling mechanisms such as DNA repair and assessment of DNA damage (check points). Thus, failure in any of these steps can cause a defective apoptotic response that plays a decisive role in both tumorigenesis and drug resistance in tumor treatment. More distinctly, the capability of cancer cells to go into apoptosis prevents further neoplastic changes. Generally, the purpose of this study is to investigate the balance between formation of genomic damage and induction of apoptosis under genotoxic stress. After genotoxic insult there are different possibilities for the fate of a cell (Figure 1). The genomic integrity is analyzed at cellular checkpoints, usually leading to a delay in cell cycle progression if DNA was damaged. Mutations in genes such as p53 and p21 change the cellular response to genotoxic stress and may alter the balance between apoptosis and genomic damage. However, p53 is usually mutated or not expressed in 70% of human tumors. Alterations in p53 states that reflect distinct apoptotic response upon induction of DNA damage were examined. In this study, three cell lines with distinct p53 states were used: TK6 harboring wild-type p53, WTK1 with mutated p53 and NH32 with knocked out p53. In the present work we applied different approaches to investigate the correlation between DNA damage and apoptotic responsiveness in cancer cell lines with different p53 states or in hormone responsive cell lines with over expressed bcl-2 gene. We were focused on effects caused by temporary down regulation of the p53 and Bcl-2 activity in human lymphoblastoid cell lines. In addition, we investigated the impact of estradiol-induced proliferation on apoptosis and DNA damage in stably transfected cells with bcl-2gene.
This study should contribute to the important field of pharmacogenetics by: firstly, establishing an easy and safe phenotyping method that combines the activity determination of all three previously mentioned CYPs (CYP2D6, CYP2C9, and CYP2C19) into one phenotyping cocktail and secondly, improving the knowledge about the predictive power of the genotype for the measured phenotype. It was indeed possible to develop a save, easy-to-use, fast and simultaneous phenotyping procedure for the important genetic polymorphic enzymes CYP2D6 and CYP2C9. To accomplish that, interaction studies with the chosen probe drugs dextromethorphan (DEX, CYP2D6), flurbiprofen (FLB, CYP2C9) and omeprazole (OME, CYP2C19) were conducted. It could be proven that DEX and FLB can be administered in combination, whereas OME alters the phenotyping results of CYP2C9. This is a new finding as in 2004 a phenotyping cocktail was published that used FLB and OME in combination. However, to our knowledge, no interaction tests were carried in that study. The new phenotyping procedure is not only verified by prior probe drug interaction studies, it also has other advantages over phenotyping cocktails found in literature. Firstly, save probe drugs are used in very small doses. This is possible due to the new sensitive LC-MS/MS methods that were evaluated. Secondly, the new phenotyping procedure is very fast and on-invasive. Urine has to be collected only for 2 h and the results also suggest that the time consuming glucuronide cleavage of the CYP2D6 dependent metabolite dextrorphan, usually carried out before CYP2D6 phenotyping, may be unnecessary. Most importantly, however, new insights into the phenotype prediction from genotype for CYP2C9 and CYP2D6 could be gained within this study. Nearly 300 phenotyped Caucasian subjects were also genotyped for the most important known variant alleles for CYP2D6, CYP2C9 and CYP2C19 using several established and newly developed genoptyping methods. Therefore, a direct correlation between phenotype and genotype could be conducted for CYP2D6 and CYP2C9. Employing linear modeling, it was possible to assign activity coefficients to each of the detected CYP2D6 and CYP2C9 alleles, thereby estimating their contribution to the resulting enzyme activity. This might facilitate the prediction of the CYP2D6 and CYP2C9 metabolic status of a subject knowing only its respective genotypes. Especially the new CYP2D6 genotype phenotype correlation model might allow for more precise phenotype prediction for the included variant alleles than was possible until now. Taken together, this study substantially contributes to the important research field of pharmacogenetics by (i) developing a save and easy-to-use phenotyping combination for CYP2D6 and CYP2C9, and (ii) by establishing activity coefficients for each of the detected CYP2D6 and CYP2C9 alleles, thereby allowing for a more precise prediction of the phenotype from genotype.
b-adrenergic receptors (b-ARs) participate strongly in the development of cardiac hypertrophy and human heart failure. Stimulation of b-adrenergic receptors with catecholamines as well as cardiac overexpression of b1-ARs or of Gas-proteins in transgenic mice induces cardiac hypertrophy. However, direct activation of their downstream targets, such as adenylyl cyclase (AC) or protein kinase A do not promote a significant degree of cardiac hypertrophy. These findings suggest that additional events may occur and that these events require Gas-protein activation. A hypertrophic pathway involving Gaq-protein coupled receptors has recently been described. Upon activation of Gaq-coupled receptors Gbg-subunits are released from Gaq and bind directly to the activated Raf/Mek/Erk cascade. Direct interaction between bg-subunits and activated Erk1/2 leads to an additional autophosphorylation of Erk2 at threonine 188, which mediates cardiac hypertrophy. Murine hearts, as well as isolated cardiomyocytes present an increase in Erk2Thr188-phosphorylation upon b-AR activation. Similarly overexpression of phosphorylation deficient Erk2 mutants (Erk2T188S and Erk2T188A) reduces b-AR mediated cardiomyocyte hypertrophy. Increase in left ventricular wall thickness, fibrosis and up-regulation of natriuretic peptide synthesis, which are physiological features for cardiac hypertrophy, are strongly inhibited in transgenic mice with a cardiac expression of Erk2T188S after two weeks of sustained isoproterenol treatment. It could further be shown in this work that b-AR mediated cardiac hypertrophy requires two distinct pathways initiated by Gs-protein activation: the canonical phosphorylation of Erk1/2 via adenylyl cyclase and the direct interaction of released bg-subunits with activated Erk1/2. Coincidence of both events leads to Erk2Thr188-phosphorylation, which activates then different transcription factors responsible for cardiac hypertrophy. Sequestration of bg-subunits by overexpression of the C-terminus of GRK2 bark-ct and inhibition of adenylyl cyclase efficiently reduced the hypertrophic response to isoproterenol, whereas direct activation of AC by forskolin failed to induce Erk2Thr188-phosphorylation and cardiomyocyte hypertrophy. These findings may help to develop new therapeutic strategies for the prevention of cardiac hypertrophy and maladaptive remodeling of the heart.
Azoles are important chemicals used as antifungal agents in agriculture and human medicine, but also as cytostatic drugs in tumour chemotherapy. Antifungal activities are based on inhibition of lanosterol-14α-demethylase (CYP51). CYP51 catalyses the oxidative removal of the methyl group # 32 of lanosterol to produce follicular fluid meiosis activating steroid (FF-MAS). For fungi the later resulting ergosterol is an essential compound of the cell membrane. Exposed fungi lack ergosterol, which leads to a collapse of the cell membrane. In mammals cholesterol, the downstream product of lanosterol-14α-demethylation necessary for the synthesis of bile acids, mineral corticoids, glucocorticoids and sex steroids, can be supplemented with food intake. However FF-MAS and the resulting T-MAS (testis meiosis activating steroids), the direct products of the CYP51 reaction, act as meiosis-activating steroids on ovaries and testes and are not supplemented with food intake. Inhibition of CYP51 in humans may therefore affect the endocrine system and is an unwanted side effect of azoles. Aromatase (CYP19) catalyses the demethylation of testosterone to estradiol and is inhibited by azoles. Reduction of estrogen levels by CYP19 inhibition is the working principle of cytostatic drugs used in breast cancer therapy but is considered an unwanted side effect for azoles used to treat fungal infections. A favourable fungicide or antifungal drug should be a strong inhibitor of fungal CYP51. In contrast human CYP51 and human CYP19 should not be inhibited by an azole fungicide or antifungal agent. The favourable cytostatic drug should show a high potency towards human CYP19. Neither human CYP51 nor fungal CYP51 should be inhibited by a cytostatic drug. The aim of this work was to assess: are fungicides and antifungal drugs strong inhibitors of fungal CYP51? In return do they not inhibit human CYP51 and human CYP19? Do cytostatic drugs strongly inhibit human CYP19? And in return do they not inhibit human CYP51 or fungal CYP51? Inhibitory potencies of 22 azole compounds used for the three purposes were tested in four inhibition assays: i) on commercially available human CYP19 utilising a fluorescent pseudo substrate dibenzylfluorescein (DBF) ii) on CYP19 utilising testosterone as substrate iii) on human CYP51 and iv) Candida albicans CYP51 utilising lanosterol as substrate. Product formation was measured by liquid chromatography – tandem mass spectrometry utilising photospray ionisation (APPI). A functional human CYP51 was available from BD Gentest Cooperation. A functional enzyme complex comprising of the Candida albicans lanosterol-14α-demethylase and the Candida tropicalis oxidoreductase was expressed in the baculovirus system. When comparing inhibitory potencies on CYP19, human CYP51 and Candida albicans CYP51 a number of agents show desirable patterns of inhibition e.g. the two cytostatic drugs, or two antifungal agents used in human medicine, fluconazole and itraconazole, and a wide variety of the fungicides, e.g. cyproconazole and hexaconazole. Undesirable patterns of inhibition were exhibited by a number of compounds, e.g. prochloraz, bifonazole, ketoconazole and miconazole. Seven compounds show a more complex picture of inhibitory potencies though. To get a picture of residue levels of azoles in food in a model case an LC-ESI-MS/MS method was developed for the determination of azole compounds in wine. All residues were below the maximum residue levels set by authorities. To classify the inhibitory potencies on the different enzyme systems IC50 values obtained were compared to exposure levels measured in farmers, maximum plasma concentrations in humans reported after exposure to antifungal drugs and to acceptable daily intake levels set by authorities. Based on the findings presented, the following conclusions can be drawn. The risk for agricultural workers set by exposure to azole fungicides with respect to human CYP51 and CYP19 can be regarded as negligible when safety measures are adhered to. As a matter of principle however, the usage of bifonazole, miconazole and ketoconazole has to be viewed with caution in respect to the high level of inhibition of human CYP51 and/or CYP19. Under the assumption that the acceptable daily intake amounts set by authorities for azole compounds are not exceeded the residues do not pose a threat to consumer safety judged by our findings. Inhibition of CYP19 with the consequence of a reduction of estradiol levels has to be regarded as a possible disrupting effect of the hormone balance. The relevance of FF-MAS and T-MAS in the endocrine system however still has to be evaluated completely bringing with it the question of how much importance has to be attached to the inhibition of human CYP51.
Adenosine receptors that belong to the rhodopsin-like G protein-coupled receptors (GPCRs) are involved in a lot of regulatory processes and are widely distributed throughout the body which makes them an attractive target for drugs. However, pharmacological knowledge of these receptors is still limited. A big advance regarding the structural knowledge of adenosine receptors was the development of the first crystal structure of the adenosine A2A receptor in 2008. The crystal structure revealed the amino acids that form the ligand binding pocket of the receptor and depicted the endpoint of receptor movement in the ligand binding process. Within the scope of this work two members of the adenosine receptor family were investigated, namely the adenosine A1 and the A2A receptor (A1R, A2AR). A1R was generated on base of the previously developed A2AR. Receptors were tagged with fluorophores, with the cyan fluorescent protein (CFP) at the C-terminal end of receptor and the Fluorescein Arsenical Hairpin binder (FlAsH) binding sequence within the third intracellular loop of receptors. Resulting fluorescent receptor sensors
A1 Fl3 CFP and A2A Fl3 CFP were investigated with help of Fluorescence Resonance Energy Transfer (FRET) measurements within living cells. FRET experiments enable the examination of alteration in the distance of two fluorophores and thus the observation of receptor dynamical movements.
For comparison of A1R and A2AR regarding receptor dynamical movement upon ligand binding, fluorescent receptor sensors A1 Fl3 CFP and A2A Fl3 CFP were superfused with various ligands and the outcomes of FRET experiments were compared regarding signal height of FRET ratio evoked by the distinct ligand that is correlated to the conformational change of receptor upon ligand binding. Beside the different direction of FRET ratio upon ligand binding at A1R and A2AR sensor, there were differences observable when signal height and association and dissociation kinetics of the various ligands investigated were compared to each other. Differences between the adenosine receptor subtypes were especially remarkable for the A1R subtype selective agonist CPA and the A2AR subtype selective agonist CGS 21680. Another part of the project was to investigate the influence of single amino acids in the ligand binding process within the fluorescent A1R sensor. Amino acid positions were derived from the crystal structure of the A2AR forming the ligand binding pocket and these amino acids were mutated in the A1R structure. Investigation of the A1R sensor and its mutants regarding confocal analysis showed involvement
of some amino acids in receptor localization. When these amino acids were mutated receptors were not expressed in the plasma membrane of cells. Some amino acids investigated were found to be involved in the ligand binding process in general whereas other amino acids were found to have an influence on the binding of distinct structural groups of the ligands investigated. In a further step, A1R and A2AR were N-terminally tagged with SNAP or CLIP which allowed to label receptor sensors with multiple fluorophores. With this technique receptor distribution in cells could be investigated with help of confocal analysis. Furthermore, ligand binding with fluorescent adenosine receptor ligands and their competition with help of a non-fluorescent antagonist was examined at the SNAP tagged A1R and A2AR. Finally the previously developed receptor sensors were combined to the triple labeled receptor sensors SNAP A1 Fl3 CFP and SNAP A2A Fl3 CFP which were functional regarding FRET experiments and plasma membrane expression was confirmed via confocal analysis. In the future, with the help of this technique, interaction between fluorescent ligand and SNAP tagged receptor can be monitored simultaneously with the receptor movement that is indicated by the distance alteration between FlAsH and CFP. This can
lead to a better understanding of receptor function and its dynamical movement upon ligand binding which may contribute to the development of new and more specific drugs for the A1R and A2AR in the future.
Conjugation of reactive intermediates of drugs with proteins or DNA may result in toxic effects such as hepatotoxicity, agranulocytosis, allergies, tumors, etc. From 1975 to 1999, 2.9% of drugs were withdrawn from the market due to such severe adverse drug reactions. Thus, formation of chemically reactive intermediates is a widely discussed problem in drug development processes. Early detection of potentially toxic compounds is required for drug discovery and drug development. Conjugation of such electrophilic compounds with glutathione (GSH) is one of the most important detoxifying reactions in vivo. Processing of these GSH-conjugates ultimately leads to the formation of renally cleared mercapturic acids, which may also be oxidized to sulfoxides. Thus, mercapturic acids may be generated and detected in vitro and non-invasively in vivo in urine to assess the reactivity of a compound in early stages of drug development processes. Therefore, the aim of this work was to develop and evaluate a HPLC-MS/MS screening method for simple and rapid detection and characterization of known and unknown mercapturic acids and application of the method to several different matrices. Based on the common constant neutral loss (CNL) of 129 Da of all mercapturic acids tested (in negative ion mode), a CNL survey scan was performed using a linear ion trap instrument and was combined with two enhanced product ion (EPI) scans with different collision energies to characterize the detected signals. The CNL resulted from the cleavage between the sulfur and the carbon atom in the N-acetyl-L-cysteine moiety. After optimization of the experimental parameters, the detection limits of the reference substances in rat urine ranged from 0.3 to 15.5 pmol on column (i.e. 20 ng/ml to 800 ng/ml). For in vitro evaluation of the method, the model compounds acetaminophen, diclofenac, bifonazole, clozapine, troglitazone, carbamazepine, and bisphenol A were screened for formation of reactive intermediates and, hence, detection of the corresponding mercapturic acids. To determine possible species- and tissue-specific toxicities, the model compounds were incubated with stimulated neutrophils and with liver microsomes from rats and humans. Species-specific differences were observed in incubations of acetaminophen and diclofenac with rat and human hepatic microsomes. Tissue-specific differences in biotransformation of the model compounds in incubations with human neutrophils and human liver microsomes were observed for diclofenac, carbamazepine, clozapine, and bifonazole. The developed HPLC-MS/MS method was also evaluated in vivo by analysis of rat and human urine. Drug-related mercapturic acids were detected in urine of rats orally treated with acetaminophen (20 mg/kg and 640 mg/kg b.w.) or diclofenac (10 mg/kg and 20 mg/kg b.w.). Human urine samples were analyzed before and after oral administration of a clinically used dose of 500 mg and 50 mg of acetaminophen. Besides detection of the mercapturic acid of N-acetylbenzoquinoneimine (AAP-MA), a second mercapturic acid with m/z 327 occurred dose-dependently in rat and human urine samples after administration of acetaminophen. Further investigations on identification of this metabolite using authentic compounds and comparing their MS/MS mass spectra demonstrated oxidation of AAP-MA to stereoisomeric sulfoxides in vivo. For diclofenac, a novel mercapturic acid with m/z 441 was detected in rat urine samples that was identical to a metabolite obtained in incubations with human neutrophils before. The in vivo formation of this diclofenac metabolite is described here for the first time. In addition, three endogenously formed mercapturic acids were detected and identified. In conclusion, the results of the in vitro and in vivo evaluation demonstrate the advantages of the rapid and generic HPLC-MS/MS screening method for the detection of mercapturic acids, that can be obtained with a minimum of sample preparation and a high throughput in diverse matrices.
Evaluation of 1H-NMR and GC/MS-based metabonomics for the assessment of liver and kidney toxicity
(2009)
For the assessment of metabonomics techniques for the early, non-invasive detection of toxicity, the nephrotoxins gentamicin (s.c. administration of 0, 60 and 120 mg/kg bw 2x daily for 8 days), ochratoxin A (p.o. administration of 0, 21, 70 and 210 µg/kg bw 5 days/week for 90 days) and aristolochic acid (p.o. administration of 0, 0.1, 1.0 and 10 mg/kg bw for 12 days) were administered to rats and urine samples were analyzed with 1H-NMR and GC/MS. Urine samples from the InnoMed PredTox project were analyzed as well, thereby focusing on 1H-NMR analysis and bile duct necrosis as histopathological endpoint. 1H-NMR analysis used water supression with the following protocol: 1 M phosphate buffer, D2O as shift lock reagent, D4-trimethylsilylpropionic acid as chemical shift reference, noesygppr1d pulse sequence (Bruker). For multivariate data analysis, spectral intensity was binned into 0.04 ppm wide bins. GC/MS analysis of urine was carried out after protein precipitation with methanol, drying, derivatization with methoxyamine hydrochloride in pyridine and with methyl(trimethylsilyl)trifluoroacetamide on a DB5-MS column using EI ionization. The chromatograms were prepared for multivariate data analysis using the R-program based peak picking and alignment software XCMS version 2.4.0. Principal component analysis (PCA) to detect and visualize time-point and dose-dependent differences between treated animals and controls and orthogonal projection to latent structures discriminant analysis (OPLS-DA) for identification of potential molecular markers of toxicity was carried out using SIMCA P+ 11.5 1H-NMR-based markers were identified and quantified with the Chenomx NMR Suite, GC/MS based markers were identified using the NIST Mass Spectral Database and by co-elution with authentic reference standards. PCA of urinary metabolite profiles was able to differentiate treated animals from controls at the same time as histopathology. An advantage over classical clinical chemistry parameters regarding sensitivity could be observed in some cases. Metabonomic analysis with GC/MS and 1H-NMR revealed alterations in the urinary profile of treated animals 1 day after start of treatment with gentamicin, correlating with changes in clinical chemistry parameters and histopathology. Decreased urinary excretion of citrate, 2-oxoglutarate, hippurate, trigonelline and 3-indoxylsulfate increased excretion of 5-oxoproline, lactate, alanine and glucose were observed. Ochratoxin A treatment caused decreased excretion of citrate, 2-oxoglutarate and hippurate and and increased excretion of glucose, myo-inositol, N,N-dimethylglycine, glycine, alanine and lactate as early as 2 weeks after start of treatment with 210µg OTA/kg bw, correlating with changes in clinical chemistry parameters and histopathology. Integration of histopathology scores increased confidence in the molecular markers discovered. Aristolochic acid treatment resulted in decreased urinary excretion of citrate, 2-oxoglutarate, hippurate and creatinine as well as increased excretion of 5-oxoproline, N,N-dimethylglycine, pseudouridine and uric acid. No alterations in clinical chemistry parameters or histopathology were noted.Decreased excretion of hippurate indicates alterations in the gut microflora, an effect that is expected as pharmacological action of the aminoglycoside antibiotic gentamicin and that can also be explained by the p.o. administration of xenobiotica. Decreased Krebs cycle intermediates (citrate and 2-oxoglutarate) and increased lactate is associated with altered energy metabolism. Increased pseudouridine excretion is associated with cell proliferation and was observed with aristolochic acid and ochratoxin A, for which proliferative processes were observed with histopathology. 5-oxoproline and N,N-dimethylglycine can be associated with oxidative stress. Glucose, a marker of renal damage in clinical chemistry, was observed for all three nephrotoxins studied. Single study analysis with PCA of GC/MS chromatograms and 1H-NMR spectra of urine from 3 studies conducted within the InnoMed PredTox project showing bile duct necrosis revealed alterations in urinary profiles with the onset of changes in clinical chemistry and histopathology. Alterations were mainly decreased Krebs cycle intermediates and changes in the aromatic gut flora metabolites, an effect that may result as a secondary effect from altered bile flow. In conclusion, metabonomics techniques are able to detect toxic lesions at the same time as histopathology and clinical chemistry. The metabolites found to be altered are common to most toxicities and are not organ-specific. A mechanistic link to the observed toxicity has to be established in order to avoid confounders such as body weight loss, pharmacological effects etc. For pattern recognition purposes, large databases are necessary.
Investigation of dynamic processes of prototypical class A GPCRs by single-molecule microscopy
(2020)
In this work, two projects were pursued.
In the first project, I investigated two different subtypes of opioid receptors, which play a key role as target for analgesia. A set of subtype specific fluorescent ligands for μ opioid receptor (MOR) and δ opioid receptor (DOR) was characterised and used to gain insights into the diffusion behaviour of those receptors. It was shown that the novel ligands hold photophysical and pharmacological properties making them suitable for single-molecule microscopy. Applying them to wild-type receptors expressed in living cells revealed that both sub-types possess a heterogeneous diffusion behaviour. Further- more, the fluorescent ligands for the MOR were used to investigate homodomerisation, a highly debated topic. The results reveal that only ≈ 5 % of the receptors are present as homodimers, and thus the majority is monomeric. G-protein coupled receptors (GPCRs) play a major role as drug targets. Accordingly, understanding the activation process is very important. For a long time GPCRs have been believed to be either active or inactive. In recent years several studies have shown, that the reality is more complex, involving more substates. [1, 2, 3, 4] In this work the α 2A AR was chosen to investigate the activation process on a single-molecule level, thus being able to distinguish also rare or short-lived events that are hidden in ensemble mea- surements. With this aim, the receptor was labelled intracellular with two fluorophores using supported membranes. Thus it was possible to acquire movies showing qualita- tively smFRET events. Unfortunately, the functionality of the used construct could not be demonstrated. To recover the functionality the CLIP-tag in the third intracellular loop was replaced successfully with an amber codon. This stop codon was used to insert an unnatural amino acid. Five different mutants were created and tested and the most promising candidate could be identified. First ensemble FRET measurements indicated that the functionality might be recovered but further improvements would be needed. Overall, I could show that single-molecule microscopy is a versatile tool to investigate the behaviour of typical class A GPCRs. I was able to show that MOR are mostly monomeric under physiological expression levels. Furthermore, I could establish intra- cellular labelling with supported membranes and acquire qualitative smFRET events.
Mammalian haloacid dehalogenase (HAD)-type phosphatases are a large and ubiquitous family of at least 40 human members. Many of them have important physiological functions, such as the regulation of intermediary metabolism and the modulation of enzyme activities, yet they are also linked to diseases such as cardiovascular or metabolic disorders and cancer.
Still, most of the mammalian HAD phosphatases remain functionally uncharacterized.
This thesis reveals novel cell biological and physiological functions of the phosphoglycolate phosphatase PGP, also referred to as AUM. To this end, PGP was functionally characterized by performing analyses using purified recombinant proteins to investigate potential protein substrates of PGP, cell biological studies using the spermatogonial cell line GC1, primary mouse lung endothelial cells and lymphocytes, and a range of biochemical techniques to characterize Pgp-deficient mouse embryos.
To characterize the cell biological functions of PGP, its role downstream of RTK- and integrin signaling in the regulation of cell migration was investigated. It was shown that PGP inactivation elevates integrin- and RTK-induced circular dorsal ruffle (CDR) formation, cell spreading and cell migration. Furthermore, PGP was identified as a negative regulator of directed lymphocyte migration upon integrin- and GPCR activation.
The underlying mechanisms were analyzed further. It was demonstrated that PGP regulates CDR formation and cell migration in a PLC- and PKC-dependent manner, and that Src family kinase activities are required for the observed cellular effects. Upon integrin- and RTK activation, phosphorylation levels of tyrosine residues 1068 and 1173 of the EGF receptor were elevated and PLCγ1 was hyper-activated in PGP-deficient cells. Additionally, PGP-inactivated lymphocytes displayed elevated PKC activity, and PKC-mediated cytoskeletal remodeling was accelerated upon loss of PGP activity. Untargeted lipidomic analyses revealed that the membrane lipid phosphatidylserine (PS) was highly upregulated in PGP-depleted cells.
These data are consistent with the hypothesis that the accumulation of PS in the plasma membrane leads to a pre-assembly of signaling molecules such as PLCγ1 or PKCs that couple the activation of integrins, EGF receptors and GPCRs to accelerated cytoskeletal remodeling.
Thus, this thesis shows that PGP can affect cell spreading and cell migration by acting as a PG-directed phosphatase.
To understand the physiological functions of PGP, conditionally PGP-inactivated mice were analyzed. Whole-body PGP inactivation led to an intrauterine growth defect with developmental delay after E8.5, resulting in a gradual deterioration and death of PgpDN/DN embryos between E9.5 and E11.5. However, embryonic lethality upon whole-body PGP inactivation was not caused by a primary defect of the (cardio-) vascular system. Rather, PGP inactivated embryos died during the intrauterine transition from hypoxic to normoxic conditions.
Therefore, the potential impact of oxygen on PGP-dependent cell proliferation was investigated. Analyses of mouse embryonic fibroblasts (MEFs) generated from E8.5 embryos and GC1 cells cultured under normoxic and hypoxic conditions revealed that normoxia (~20% O2) causes a proliferation defect in PGP-inactivated cells, which can be rescued under
hypoxic (~1% O2) conditions. Mechanistically, it was found that the activity of triosephosphate isomerase (TPI), an enzyme previously described to be inhibited by phosphoglycolate (PG) in vitro, was attenuated in PGP-inactivated cells and embryos. TPI constitutes a critical branch point between carbohydrate- and lipid metabolism because it catalyzes the isomerization of the glycolytic intermediates dihydroxyacetone phosphate (DHAP, a precursor of the glycerol backbone required for triglyceride biosynthesis) and glyceraldehyde 3’-phosphate (GADP).
Attenuation of TPI activity, likely explains the observed elevation of glycerol 3-phosphate levels and the increased TG biosynthesis (lipogenesis). Analyses of ATP levels and oxygen consumption rates (OCR) showed that mitochondrial respiration rates and ATP production were elevated in PGP-deficient cells in a lipolysis-dependent manner. However under hypoxic conditions (which corrected the impaired proliferation of PGP-inactivated cells), OCR and ATP production was indistinguishable between PGP-deficient and PGP-proficient cells. We therefore propose that the inhibition of TPI activity by PG accumulation due to loss of PGP activity shifts cellular bioenergetics from a pro-proliferative, glycolytic metabolism to a lipogenetic/lipolytic metabolism.
Taken together, PGP acts as a metabolic phosphatase involved in the regulation of cell migration, cell proliferation and cellular bioenergetics. This thesis constitutes the basis for further studies of the interfaces between these processes, and also suggests functions of PGP for glucose and lipid metabolism in the adult organism.