Refine
Year of publication
- 2007 (79) (remove)
Document Type
- Doctoral Thesis (79) (remove)
Language
- English (79) (remove)
Has Fulltext
- yes (79)
Is part of the Bibliography
- no (79) (remove)
Keywords
- Carrier-Proteine (2)
- Cytologie (2)
- Histologie (2)
- Membranproteine (2)
- Molekülstruktur (2)
- NMR-Spektroskopie (2)
- Proteinfaltung (2)
- Quantenchromodynamik (2)
- ABC-Transporter (1)
- Ackerschmalwand (1)
Institute
- Biochemie und Chemie (23)
- Biowissenschaften (15)
- Medizin (12)
- Physik (12)
- Wirtschaftswissenschaften (5)
- Frankfurt Institute for Advanced Studies (FIAS) (3)
- Geowissenschaften (3)
- Pharmazie (3)
- Geographie (1)
- Mathematik (1)
Intrinsic response properties of auditory thalamic neurons in the Gerbil (Meriones unguiculatus)
(2007)
Neurons in the medial geniculate body (MGB) have the complex task of processing the auditory ascending information from the periphery and a more extensive descending input from the cortex. Differences in the pattern of afferent and efferent neuronal connections suggest that neurons in the ventral and dorsal divisions of the MGB take different roles in this complex task. The ventral MGB (vMGB) is the primary, tonotopic, division and the dorsal MGB (dMGB) is one of the higher order, nontonotopic divisions. The vMGB neurons are arranged tonotopically, have sharp tuning properties, and a short response delay to acoustic stimuli. The dMGB neurons are not tonotopically arranged, have broad tuning properties, and a long response delay to acoustical stimuli. These two populations of neurons, with inherently different tasks, may display differences in intrinsic physiological properties, e.g. the capacity to integrate information on a single cell level. Neurons of the ventral and dorsal divisions of the MGB offer an ideal system to explore and compare the intrinsic neuronal properties related to auditory processing. Coronal slices of 200 μm thicknesses were prepared from the thalamus of 4 - 5 week old gerbils. The current-clamp configuration of the patch-clamp technique was used to do experiments on the dorsal and ventral divisions of the medial geniculate body. Slices were subsequently Nissl stained to verify the location of recording. Recordings from the dorsal and ventral divisions exhibited differences in response to depolarizing current injections. The ventral division responded with significantly shorter first spike latency (vMGB = 41.50 ± 7.7, dMGB = 128.43 ± 16.28; (p < 0.01)) and rise time constant (vMGB = 6.95 ± 0.90, dMGB = 116.67 ± 0.13; (p < 0.01)) than the dMGB. Neurons in the dorsal division possessed a larger proportion of slowly accommodating neurons (rapidly accommodating: vMGB: 89%, dMGB: 64%), including a subpopulation of neurons that fired at resting membrane potential. Neurons in the vMGB are primarily responsible for relaying primary auditory input. Dorsal MGB neurons relay converging multimodal input. A comparative analysis with the primary auditory neurons, the Type I and Type II spiral ganglion neurons, reveals a similar pattern. Type I neurons relay primary auditory input and exhibit short first spike latencies and rise time constants. The Type II neurons relay converging input from many sources, while possessing significantly slower response properties and a greater subpopulation of slowly accommodating neurons. Hence, accommodation, first spike latency, and rise time constant are suggested to be a reflection of the amount of input that must be integrated before an action potential can be fired. More converging input correlates to slower accommodation, a longer first spike latency and rise time. Conversely, a greater capacity to derive discrete input is associated with rapid accommodation, along with a short first spike latency and rise time.
The work presented in this thesis addresses a key issue of the CBM experiment at FAIR, which aims to study charm production in heavy ion collisions at energies ranging from 10 to 40 AGeV . For the first time in this kinematical range, open charm mesons will be used as a probe of the nuclear fireball. Despite of their short decay length, which is typically in the order of few 100 µm in the laboratory frame, those mesons will be identified by reconstructing their decay vertex.
This dissertation contains five independent chapters dealing with wage dispersion and unemployment. The first chapter deals with the explanation of international changes in wage inequality and unemployment in the 80s and 90s. Both theoretically and empirically, social benefits and its link to average income are blamed for the different experiences across countries. The second chapter discusses the search framework, to explain residual wage inequality and finds that institutional wage compression has ambiguous effects on employment. In the third chapter, we apply the theory to German data. We show that job-to-job transitions are important in explaining both frictions and career advances. In the fourth chapter, we empirically assess the relationship between wage dispersion and unemployment for homogeneous workers. We find that neither a frictional nor a neo-classical view in explaining this relationship are convincing. Unemployment within cells is not negatively correlated with wage dispersion. Finally, the last chapter builds a theoretical model which treats heterogeneous individuals in a production function framework and a frictional labor market. The model generates both wage dispersion within and between skill groups and both frictional and structural unemployment. In sum, the dissertation stresses the importance of modelling frictions to understand different types of wage inequality and unemployment.
Spatio-temporal dynamics of primary lymphoid follicles during organogenesis and lymphneogenesis
(2007)
Primary lymphoid follicles are structures which are important for adaptive immune responses in mammals. Within the follicles follicular dendritic cells (FDC) are maintained by constant stimuli provided by B cells. It is thought that the FDC are important for immune response. It is of interest to know how lymphoid follicles are regulated in order to understand their role in various autoimmune diseases in which these follicles are created ectopically. With the help of a tissue simulation relying on an agent-based cell model on top of a regular triangulation various scenarios suggested by the available experimental data have been investigated. In order to cope with the complexity in the simulation of immune tissue the regular triangulation has been implemented for the use on parallel computers. The algorithms for kinetic and dynamic regular triangulation have been created newly. Also the cell model underlying the simulation has been designed newly in many aspects. The simulations allowed to identify common factors that regulate the formation of lymphoid follicles normally during organogenesis in development and lymphneogenesis in the course of diseases. The generation of FDC from local stromal populations under the influence of B cell aggregates is shown to be possible with the given experimental parameters. The sequence of the organogenesis and lymphneogenesis can be described with regard to the morphology of the B and T zone. Tests for the stability of the primary lymphoid follicle system constraints the regulation of the B cell efflux. The required lymphatic vessels around the lymphoid follicle are shown to be negatively correlated with the FDC network. Moreover it is shown that the adjacent T zone consisting of its own stromal population and T cells has similar regulation principles. This easily explains the intermediate ring of B cells found around the T zone during development and certain signaling molecule deficiencies. A major result of this thesis is that the generation of FDC needs negative regulation while a number of other possible mechanisms is incompatible with the available experimental data. Moreover the observed microanatomy was brought into a functional relationship with data on the cellular level finally culminating in the proposal of new experiments that shed light on the dynamics of the primary lymphoid follicle. One conclusion is that the FDC directly or indirectly influence the angiogenesis and lymphangiogenesis processes in secondary lymphoid tissues. The work presented here may help to guide experiments with the help of computers in order to reduce the amount of experiments and design them in a way to maximize the amount of information about biological systems.
Rhythmic changes in environmental lighting conditions have ever been the most reliable environmental cue for life on earth. Nature has therefore selected a genetically encrypted endogenous clock very early in evolution, as it provided cells and subsequently organisms with the ability to anticipate persevering periods of light and darkness. Rhythm generation within the mammalian circadian system is achieved by clock genes and their protein products. The mammalian endogenous master clock, which synchronizes the body to environmental time, is located in the suprachiasmatic nucleus (SCN) of the hypothalamus. As an integral part of the time-coding system, the pineal gland serves the need to tune the body to the temporal environment by the rhythmic nocturnal synthesis and immediate release of the hormone melatonin. In contrast to the transcriptional regulation of melatonin synthesis in rodents, a post-translational shaping is indicated in the human pineal gland. Another important mediator of circadian time and seasonality to the body is the pituitary gland. The aim of this work was to elucidate regulation of melatonin synthesis in the human pineal gland. Furthermore, presence and regulation of clock genes in the human pineal and pituitary gland, and in the SCN were analyzed. Therefore, human tissue, taken from regular autopsies, was analyzed simultaneously for different parameters involved in melatonin biosynthesis and circadian rhythm generation. Presented data demonstrate that post-mortem brain tissue can be used to detect the remnant profile of pre-mortem adaptive changes in neuronal activity. In particular, our results give strong experimental support for the idea that transcriptional mechanisms are not dominant for the generation of rhythmic melatonin synthesis in the human pineal gland. Together with data obtained for clock genes and their protein products in the pituitary, data presented here offer 1) a new working hypothesis for post-translational regulation of melatonin biosynthesis in the human pineal gland, and 2) a novel twist in the molecular competence of clock gene proteins, achieved by nucleo-cytoplasmic shuttling in neuronal and neuroendocrine human tissue. Furthermore, in this study, oscillations in abundance of clock gene proteins were demonstrated for the first time in the human SCN.
The removal of apoptotic cells (AC) can be regarded as an integral component of the program to terminate inflammation. Clearance of AC by professional phagocytes such as macrophages induces an anti-inflammatory phenotype in the latter ones. Anti-inflammatory or M2 polarization is also observed in macrophages infiltrating certain human tumors. These tumor-associated macrophages (TAM) contribute actively to tumor progression by promoting immune evasion, angiogenesis and tumor cell survival. The aim of my Ph.D. thesis was to approach the mechanisms as well as the characteristics of macrophage phenotype alterations induced by AC, and to elucidate a possible connection between tumor cell apoptosis and TAM generation. In the first part of my studies, I investigated the impact of AC on macrophage viability. I could show that macrophage survival against pro-apoptotic agents increased after the interaction with AC. Protection of macrophages against cell death required activation of phosphatidylinositol-3 kinase (PI3K), extracellular signal-regulated kinase 1/2 (ERK1/2) and Ca2+ signaling, and correlated with Bcl-XL and Bcl-2 up-regulation as well as Ser136-Bad phosphorylation. Unexpectedly, neither phagocytosis nor binding of apoptotic debris to the phagocyte was necessary to induce protection. AC released the bioactive lipid sphingosine-1-phosphate (S1P), dependent on sphingosine kinase (SphK) 2, as a survival messenger. These data indicated an active role of AC in preventing cell destruction in their neighborhood. My next aim was to elucidate the mechanism of S1P production by AC. During cell death, SphK 2 was cleaved at its N-terminus by caspase-1. Thereupon, the truncated but enzymatically active fragment of SphK 2 was released from cells. This release was coupled to phosphatidylserine exposure, a hallmark of apoptosis and a crucial signal for the phagocyte/apoptotic cell interaction. Thus, I observed a link between common signaling events during apoptosis and the extracellular production of S1P, which is known to affect immune cell attraction and polarization as well as angiogenesis in cancer. In the next part of my studies, I asked for a correlation between tumor cell apoptosis and TAM polarization. During co-culture of human macrophages with human breast cancer carcinoma cells (MCF-7), the latter ones were killed, while macrophages acquired an alternatively activated phenotype. This was characterized by decreased tumor necrosis factor (TNF)-α; and interleukin (IL)-12-p70 production, but increased formation of IL-8 and IL-10. Alternative macrophage activation required tumor cell death, because a co-culture with apoptosis-resistant colon carcinoma cells (RKO) or Bcl-2-overexpressing MCF-7 cells failed to induce phenotype alterations. These phenotype alterations were also achieved with conditioned media from apoptotic tumor cells, which again argued for a soluble factor being involved. Knock-down of SphK2, but not SphK1, to attenuate S1P formation in MCF-7 cells, repressed the otherwise observed alternative macrophage polarization during co-culture. Furthermore, macrophage polarization achieved by tumor cell apoptosis or substitution of authentic S1P was characterized by suppression of pro-inflammatory nuclear factor (NF)-κB DNA binding. These findings suggested that tumor cell apoptosis-derived S1P contributes to the macrophage polarization present in human tumors. To validate these in vitro data, I used an in vivo tumor model to clarify the relevance of SphK2 and S1P in tumor development. The growth of, as well as blood vessel infiltration into SphK2 knock-down MCF-7 (MCF-7-siSphK2) xenografts in nude mice was markedly decreased in comparison to control MCF-7 xenografts. In contrast, macrophage infiltration was similar or even more pronounced. These data provided a first hint for an in vivo role of SphK2-derived S1P in macrophage polarization associated with tumor promotion. In summary, these data indicate a new mechanism how AC themselves shape macrophage polarization, which results in the termination of inflammatory responses and macrophage survival. Furthermore, my studies present evidence that human tumors may utilize this mechanism to foster growth via increased angiogenesis.
The high energy loss of heavy ions in matter as well as the small angular scattering makes heavy ion beams an excellent tool to produce almost cylindrical and homogeneously excited volumes in matter. This aspect can be used to pump short wavelength lasers. In an experiment performed at the GSI (Gesellschaft für Schwerionenforschung, Darmstadt, Germany) ion accelerator facility in December 2005 the well-known KrF* excimer laser was pumped with an intense high energy uranium beam. Pulses of an uranium beam with initial particle energy of 250 MeV per nucleon, provided by heavy-ion-synchrotron SIS-18, were delivered to the HHT-target station and then stopped inside a gas laser cell. The maximum beam intensity reached in the experiment was 2,5·109 particles per pulse, which resulted in 34 J/g specific energy deposited in the laser gas. By applying electron cooling and a bunch compression technique at SIS-18, the beam pulses were compressed down to 110 ns (FWHM). A mixture of an excimer laser premix gas (95,5% Kr + 0,5% F2) and a buffer gas (Ar 4.8) was used as the laser gas in proportions of 35/65 and 60/40, respectively. The gas pressure inside the laser cell was varied in the range of 1,2÷2 bar in continues flow mode. The experimental setup consisted of a 1 m long stainless steel tube with a number of diagnostic viewports and two mirror adjustment units. The optical cavity was formed by a flat, Alcoated mirror at the beam entrance and a second dielectrically coated, highly reflective mirror with 3 m radius of curvature at a distance of 1,3 m. A beam of heavy ions has been used to pump a short wavelength gas laser for the first time. Laser effect on the KrF* laser transition (λ = 248 nm) has been successfully demonstrated. Laser threshold for this specific setup was reached with a beam intensity of 1,2·109 particles per pulse. Laser action has been clearly proofed by the following methods: appearance of the laser line, spectral narrowing of the laser line, temporal narrowing of the laser signal, non-linear response of the laser output intensity on the pumping power, and cavity disalignment effect. An energy of the laser pulse of about 2 mJ was measured for an ion beam intensity of 2·109 particles per pulse. The time delay of the onset of the laser emission with respect to the pumping pulse was measured as a function of ion beam intensity. The dependence of spontaneous emission spectra on the gas pressure in a range of 1,3÷2 bar was observed and the optimal gas pressure for laser experiments in the sense of laser efficiency was concluded. As a next step in studying short wavelength lasers pumped with heavy ion beams it is planned to reduce the laser wavelength down to the VUV region of the spectrum, and to proceed to the excimer lasers of the pure rare gases: Xe2 * (λ = 172 nm), Kr2 * (λ = 146 nm), Ar2 * (λ = 126 nm), Ne2 * (λ = 83 nm) and He2 * (λ = 80 nm). We believe that the use of heavy ion beams as a pumping source may lead to new pumping schemes on the higher lying level transitions and considerably shorter wavelengths (XUV and X-ray spectral region), which rely on the high cross sections for multiple ionization of the target species.
Imatinib (GleevecTM; GlivecTM; formerly STI571), a specific inhibitor of Abl tyrosine kinase, is efficacious in treating Philadelphiachromosomepositive (Ph+) leukaemias such as chronic myeloid leukaemia (CML) and Ph+ acute lymphoblastic leukaemia (ALL) (Ottmann, Druker et al. 2002). Within a few years of its introduction to the clinic, Imatinib had dramatically altered the firstline therapy for CML, because it was found that most newly diagnosed CML patients in the chronic phase achieve durable responses when treated with Imatinib (Goldman and Melo 2003). However, a small percentage of these patients, as well as most advancedphase CML and Ph+ ALL patients, relapse on Imatinib therapy (Yokota, Kimura et al. 2006). Several mechanisms of refractoriness and relapse have been reported. These include point mutations within the Abl kinase domain, overexpression of BcrAbl mRNA (Hofmann, Jones et al. 2002), decreased intracellular drug levels mediated by Pglycoprotein (Pgp) (Hegedus, Orfi et al. 2002), and nonBcrAbl dependent mechanisms (activation of the SFKs) (Donato, Wu et al. 2003). In this research work, a possible means of overcoming resistance to Imatinib by the use of the specific dual Src/Abl kinase inhibitor AZD0530 has been investigated. The efficacy of AZD0530 in the treatment of Ph+ leukaemias, sensitive to or resistant to Imatinib, has been tested on cell lines, primary patient material and in vivo in transduction/transplantation mouse model of Imatinib sensitive or resistant BcrAbl dependent CML-like disease. Data with AZD0530 has been compared to cells treated with Imatinib. The potential of inhibiting both Src and Abl kinases while inducing growth arrest and apoptosis has been analysed. AZD0530 specifically inhibited the growth of CML and Ph+ ALL cells in a dosedependent manner, but has shown a marginal effect on Ph- ALL cells. Treatment of p185BcrAbl expressing Ba/F3 cells with AZD0530 has led to apoptosis induction and growth inhibition in these cells, while the untransformed Ba/F3 cells have remained unaffected. Resistance to Imatinib due to mutation in the Ba/F3MutY253F cells has been overcomed by this compound. The growth inhibitory effect of AZD0530 correlates with its induction of apoptosis. Combination of AZD0530 and Imatinib at low concentrations has shown an additive effect on the inhibition of proliferation of BV173 cells. The growth inhibition and apoptosis induction by AZD0530 have shown to be uncoupled to major changes in cell cycle. An exception is the CML blast crisis cell line BV173 which has shown a considerable G0/G1 arrest in the presence of AZD0530 and Imatinib as single agents. Immunoblotting of whole cell lysates from Imatinib or AZD0530 treated BV173, Ba/F3 expressing p185(BcrAbl) MutT253F cells and the WTSupB15 cells, for Src and BcrAbl clearly demonstrates that there is an ongoing transphosphorylation taking place between the SFKs and BcrAbl. This transphosphorylation synergizes and influences the aggressive nature of CML blast crisis and Ph+ ALL. Investigations have been carried out on downstream signaling events to determine how Src family members contribute to BcrAbl signaling. Specifically, Stat, Erk and PI3K/ Akt activation status have been characterised in Imatinib sensitive and resistant Ph+ cells. AZD0530 has significantly downregulated the activation of survival signaling pathways as shown by it’s inhibition of Stat5, Akt and Erk kinases in Ph+ cells, resistant or sensitive to Imatinib. The only exception to this has been the Imatinib resistant cell line RTSupB15, in which activated Akt kinase level has remained unaffected. AZD0530 has shown to be efficient in the treatment of cells isolated from three Ph+ leukaemic patients (resistant or sensitive to Imatinib), and has led to an induction of apoptosis. Equally, in the same patients, growth and survival pathways have been inhibited in vitro in the presence of AZD0530. An overall therapeutic effect of AZD0530 in vivo has been studied in mouse model of Imatinib sensitive and Imatinib resistant, BcrAbldependent desease. Mice with a BcrAbllike disease responded to Imatinib treatment but not to AZD0530. Using the CFU assay, an influence on the differentiation status of primary leukaemic blast stem cells have been tested. The in vivo studies as well as the CFU results have shown discrepancies to the effects of AZD0530 tested so far in this research work. These discrepancies have paralleled with the upregulation of BcrAbl in most AZD0530 treated cells. These are to be further analysed. These data elucidate the role of Src kinases in BcrAbl leukaemogenesis. Results gotten from this research work has shown that AZD0530 targets both Src and BcrAbl kinase activity and reduces the transforming potential of BcrAbl. It also shows that there is an ongoing transphosphorylation between SFKs and BcrAbl kinase. AZD0530 has proven effective in CML cell lines, Ph+ ALL cell lines and patient cells resistant to Imatinib. These have demonstrated that AZD0530 is a potential drug target which can be used to overcome Imatinib resistance.
Transcranial magnetic stimulation (TMS) is a non-invasive technique which can be used to study different intracortical excitatory and inhibitory neuronal circuits in the intact human being. In the primary motor cortex, there are essentially three different TMS measures of inhibitory neuronal circuits as determined by paired-pulse TMS: short-interval intracortical inhibition (SICI), long-interval intracortical inhibition (LICI) and interhemispheric inhibition (IHI). It was hypothesized that SICI is a GABAA receptor mediated inhibition (Ilic et al., 2002) whereas LICI and IHI are mediated by GABAB receptors (Daskalakis et al., 2002; McDonnell et al., 2006). Additionally, it was shown that these inhibitory circuits interact negatively, possible due to presynaptic GABAB receptor mediated inhibition (Sanger et al., 2001; Daskalakis et al., 2002). Which neuronal populations exactly underlie SICI, LICI and IHI, is not completely clear and by which mechanism these inhibitory circuits interact has never been tested pharmacologically so far. Thus, the effects of a single oral dose of Diazepam (DZP), a specific positive allosteric modulator at the GABAA receptor, and of Baclofen (BAC), a specific GABAB receptor agonist, on SICI, LICI and IHI as well as their interactions were tested here in a randomized, placebo controlled, double-blinded crossover study. SICI significantly increased after intake of DZP whereas BAC did not change SICI. Conversely, LICI significantly increased after intake of BAC but did not change after intake of DZP. IHI showed only a trend towards a decrease after intake of DZP but no change after intake of BAC. The interactions IHI-SICI, LICI-IHI and LICI-SICI were all negative at baseline. SICI and IHI were partially suppressed in the presence of IHI and LICI, respectively, and SICI in the presence of LICI was almost completely blocked. BAC did not change any of these interactions, whereas DZP significantly increased SICI in the presence of LICI. This study is the first to examine by means of pharmacological testing the complex interactions between different inhibitory circuits in the human motor cortex. The effects of DZP and BAC on SICI and LICI confirmed the notion that SICI is a GABAA receptor mediated intracortical inhibition whereas LICI depends on GABAB receptor mediated neurotransmission. The pharmacology of IHI at short interstimulus intervals of < 20 ms (12 ms in this study) remains still inconclusive and warrants further investigation. Findings further suggest that SICI, LICI and IHI represent three different inhibitory neuronal circuits which can be tested non-invasively by means of paired-pulse TMS. Furthermore, the data support the idea that the negative interactions IHI-SICI, LICI-IHI and LICI-SICI are most likely due to presynaptic GABAB receptor mediated autoinhibition.
Colorectal cancer is one of the most cause of cancer and death in Western societies. Recently, histone deacetylase inhibitors (HDIs), which regulate transcription through modification of chromatin structure, received considerable interest on the ground of they ability to stop the growth and induce cell death in colon cancer tumours, representing a promising transcriptional cancer therapy. This kind of cancer initiates with an activating mutation in the Wnt cascade, allowing the nuclear import of ß-catenin binding to LEF/TCF. This induces the overexpression of growthpromoting oncogenes affecting the cell cycle arrest, lineage-specific cell differentiation and apoptosis processes. In addition, ß-catenin also participates in cell-cell adhesion via interactions with E-cadherin, which can be repressed by families of transcription factors Snail and ZEB. This, and gain of vimentin has been closely correlated with local invasion and metastasis since they avoid the induction of apoptosis through the loss of cell anchorage, a phenomenon called anoikis. In this process the inactivation of the kinases Src an FAK provoking disruption of focal adhesion complexes through is involved. LAQ824 is a HDAC inhibitor derivative of hydroxamic acid, which present antitumor effect in colon and other cancer cells. The aim of this study is to analyse the effect of LAQ824 in cell proliferation, apoptosis, motility and tumour invasion in a colon carcinoma model based on the adenoma-carcinoma sequence descrying trough which pathways LAQ824 is able to cause these effects. Here I demonstrate for the first time that a HDAC inhibitor, LAQ824, induces detachmentinduced cell death of colon cancer cell lines HCT116 and HT-29, a phenomenon called anoikis, in a caspase-dependent and p53-independent manner. In this process the component of the Wnt signalling pathway ß-catenin is involved. Furthermore LAQ824 upregulates the adhesion molecule E-cadherin expression in these cell lines independently of its repressor Snail, but probably mediated by the repressor ZEB. In addition LAQ824-induced anoikis is caused by disruption of focal adhesion complexes through inhibition of the activity of the kinases FAK and Src inhibiting cell motility indicating a strong antimetastatic potential for LAQ824.
Prostaglandin D2 (PGD2) is involved in a variety of physiological and pathophysiological processes, but its role in fever is poorly understood and the data obtained so far are rather controversial. Here we investigated the effects of central PGD2 delivery and of systemic prostaglandin D synthase (PGDS) or cyclooxygenase (COX) inhibition on core body temperature (TC) and on prostaglandin levels in the cerebrospinal fluid (CSF) of rats. Both PGE2 and PGD2 were detectable in CSF samples from control rats (6.2 ± 1.1 and 17.3 ± 3.1 pg/ml, respectively). Lipopolysaccharide (LPS) injection (50 μg i.p.) induced fever during the 5-hour observation period. Five hours after LPS injection, the levels of PGE2 and PGD2 were increased in the CSF about 90-fold (541.0 ± 47.5 pg/ml) and 5-fold (95.4 ± 23.1 pg/ml), respectively. Administration of PGD2 (50 - 500 ng) into the cisterna magna (i.c.m) evoked a delayed fever response in a dose-dependent manner that was accompanied by increased levels of PGE2 in the CSF. RT-PCR analyses revealed that the increased levels of PGE2 after PGD2 administration were not caused by up-regulation of COX-2 or microsomal prostaglandin E synthase 1 (mPGES-1) in the hypothalamus. Interestingly, i.c.m. pretreatment of animals with PGD2 considerably sustained the pyrogenic effects of i.c.m. administered PGE2. Pretreatment with a novel PGDS inhibitor, EDJ300520 (10 – 40 mg/kg p.o.), 1 h prior to the LPS injection impaired the LPS-induced increase of both PGD2 and PGE2 in the CSF and inhibited the fever response. In contrast, administration of EDJ300520 3 h after LPS injection did not ameliorate the LPS-induced fever. Accordingly, the concentration of PGE2 in the CSF was not decreased after EDJ300520 treatment. However, the CSF levels of PGD2 were reduced after administration of a high dose of EDJ300520 (40 mg/kg). We also investigated the effects of antipyretic drugs on the CSF levels of PGE2 and PGD2 during LPS-induced fever. Four antipyretic drugs with different mechanisms of action were used, including ibuprofen (5 - 20 mg/kg), celecoxib (10 - 50 mg/kg), SC560 5 - 20 mg/kg), and paracetamol (50 - 150 mg/kg). Each drug was used in three different doses and was orally administered 3 h after the LPS injection. All drugs were capable to attenuate the LPS-induced fever. The decrease of TC paralleled the reduction of PGE2 levels in the CSF. Of note, there was a tendency to reduced PGD2 levels in the CSF after treatment with the antipyretic drugs. However, only SC560 and the high dose of celecoxib (50 mg/kg) reduced the PGD2 levels significantly. In summary, our experiments underscore the pivotal role of PGE2 as the principal downstream mediator of fever. Moreover, we demonstrate that PGD2 is also involved in the mechanisms underlying fever. Our data suggest that PGD2 exerts an indirect pyrogenic effect by modulating the availability of PGE2 in the CSF. Additional studies are needed to explore the exact mechanism by
1. Halobacillus halophilus akkumuliert zum Ausgleich geringer, extrazellulärer Wasserpotentiale kompatible Solute. Bei Anzuchten in Gegenwart von 0,4 – 1,5 M NaCl wurden Glutamin und Glutamat als die dominierenden kompatiblen Solute identifiziert, während zwischen 2,0 und 3,0 M NaCl Prolin das dominierende Solut darstellt. Außerdem wurde Ectoin als zweites kompatibles Solut gefunden, das spezifisch bei hohen Salzgehalten akumuliert wird. Die Konzentrationen während der exponentiellen Wachstumsphase war jedoch um den Faktor 6 – 7 geringer im Vergleich zu Prolin. 2. Aus Wachstumsexperimenten in Gegenwart unterschiedlicher Anionen war bekannt, dass Glutamat, im Gegensatz zu Gluconat und Nitrat, in der Lage ist, das Wachstum von H. halophilus auch in Abwesenheit von Chlorid zu ermöglichen. Um der Frage nachzugehen, ob die wachstumsfördernde Wirkung von unphysiologisch hohen Glutamat-Konzentrationen im Medium auf die Verwendung von Glutamat als kompatiblem Solut in den Zellen zurückzuführen ist, wurden Gesamtsolutepools von Chlorid-, Nitrat-, Gluconat- und Glutamat-gezogenen Zellen gemessen. In NaCl-gezogenen Zellen zeigte sich Glutamat als dominantes Solut, während Prolin und Glutamin einen geringeren Teil am Gesamtpool ausmachten. In Nitrat-gezogenen Zellen betrug der Gesamtpool nur noch 83% und in Gluconat-gezogenen Zellen nur noch 27% im Vergleich zu Chlorid-gezogenen Zellen. Zellen, die mit Glutamat gezogen wurden, zeigten jedoch eine Gesamtkonzentration an Soluten, die ca. 100% über dem Vergleichswert aus Chlorid-gezogenen Zellen lag. Die Konzentration an Glutamin in den Zellen stieg dabei um 168%, die Konzentration an Glutamat sogar um 299%. Die Prolinkonzentration verringerte sich um 32%. Diese Daten belegen, dass der wachstumsstimulierende Effekt von Glutamat auf die Verwendung als kompatibles Solut zurückzuführen ist. 3. Zur Untersuchung der molekularen Grundlage der Salzadaptation sowie der Abhängigkeit von Chlorid in H. halophilus wurde in Zusammenarbeit mit der Gruppe von Prof. D. Oesterhelt (MPI für Biochemie, Martinsried) die Sequenzierung des Genoms begonnen. Das Projekt ist zur Zeit noch nicht abgeschlossen und befindet sich in der „Lückenschluß-Phase“. Die bisherigen Sequenzdaten konnten dennoch für die in dieser Arbeit beschriebenen Untersuchungen herangezogen werden. Das Genom besitzt eine Größe von ca. 4,1 Mbp mit einem ungefähren GC-Gehalt von 40%. Außerdem wurden 2 Plasmide identifiziert mit einer Größe von 16047 und 3329 bp. 4. Die Schlüsselgene bekannter Biosynthesewege für Glutamin und Glutamat konnten identifiziert werden. Darunter befinden sich zwei Isogene für eine Glutamatdehydrogenase (gdh1 und gdh2), ein Gen für die große Untereinheit einer Glutamatsynthase (gltA), zwei Gene für die kleine Untereinheit einer Glutamat-Synthase (gltB1 und gltB2) und zwei Isogene für eine Glutaminsynthetase (glnA1 und glnA2). glnA1 befindet sich in einem Cluster zusammen mit einem Gen, das für einen Regulator kodiert (glnR), wie er auch aus B. subtilis bekannt ist. Über reverse Transkription von mRNA und anschließender PCR-Analyse konnte gezeigt werden, dass sowohl gltA/gltB1 als auch glnA1/glnR in einem Operon organisiert sind. 5. Wurde die Transkriptmenge der in Punkt 4 erwähnten Biosynthesegene in Zellen quantifiziert, die in Gegenwart unterschiedlicher Salzkonzentrationen (0,4 – 3,0 M NaCl) gezogen wurden, so zeigte sich keine Abhängigkeit von der Salzkonzentration für die Gene gltA, glnA1 und gdh1. Über die Transkriptmengen von gdh2 ließ sich keine abschließende Aussage treffen, da die gefundenen Transkriptmengen sehr gering waren und daher zu sehr großen Varianzen bei der Quantifizierung führten. Eine klare Abhängigkeit der Transkriptmenge von der im Medium zugesetzten Salzkonzentration konnte für glnA2 gezeigt werden. Die glnA2 mRNA-Menge stieg dabei mit steigender Salzkonzentration an und erreichte bei 1,5 – 2.0 M NaCl ein Maximum. Bei diesen Salzkonzentrationen war die Menge an mRNA ca. 4 mal höher als der Vergleichswert bei 0,4 M NaCl. Bei höhern Salzkonzentrationen sank die Menge an Transkript wieder leicht und war dann ca. nur noch 3 mal so hoch wie bei 0,4 M NaCl. 6. Die zelluläre Konzentration der glnA2-Transkripte in Abhängigkeit unterschiedlicher Anionen im Anzuchtmedium wurde untersucht. Die Quantifizierung der glnA2–mRNA ergab eine 2 mal höhere Transkriptmenge in Gegenwart von Chlorid verglichen mit Nitrat oder Gluconat. 7. Es wurde nach Enzymaktivitäten der bekannten Schlüsselenzyme im Glutamat und Glutamin-Biosyntheseweg gesucht. Eine Glutamatdehydrogenase und eine Glutamatsynthase – Aktivität konnte nicht oder nur in vernachlässigbarem Maße nachgewiesen werden. Im Gegensatz dazu konnt eine Glutaminsynthetase – Aktivität eindeutig belegt werden. Diese Aktivität erwies sich abhängig von der Art und der Konzentration des angebotenen Anions im Medium. Maximale Aktivitäten wurden mit NaCl in einer Konzentration von 2,5 – 3,0 M erreicht. Interessanterweise erwies sich die Glutaminsynthetase – Aktivität auch abhängig von der Art des im Testpuffers verwendeten Anions. Hier zeigte sich eine deutliche Stimulierung der Aktivität durch das Anion Chlorid. [Die für diesen Punkt zugrunde liegenden Daten wurden im Rahmen einer von mir mitbetreuten Diplomarbeit von Jasmin F. Sydow erhoben und sind aus Gründen der vollständigen Darstellung des Projektverlaufes mitaufgeführt!] 8. Wie im Punkt 1 dargelegt, wird Prolin vor allem bei hohen Salzkonzentrationen in H. halophilus - Zellen akkumuliert. Neben der Abhängigkeit von der Salzkonzentration wurde außerdem die Abhängigkeit von der Wachstumsphase untersucht. Die Analyse der Prolinkonzentrationen während verschiedener Wachstumsphasen in Kulturen, die bei 1,0 bzw. 2,5 M NaCl angezogen wurden, zeigte, (i) dass die Prolinkonzentration während der frühen exponentiellen Phase ca. 2,5-fach erhöht war im Vergleich zu Niedrigsalz-Zellen, (ii) dass die Prolinkonzentration beim Übergang von der frühen in die späte exponentielle Phase dramatisch abnahm (um 64% bei 2,5 M NaCl) und dass (iii) in der stationären Phase Prolin praktisch nicht mehr nachzuweisen war. 9. Die Biosynthesegene für die Herstellung von Prolin aus Glutamat konnten im Genom von H. halophilus identifiziert werden. Es handelt sich dabei um ein Cluster von 3 Genen, die für eine putative Pyrrolin-5-carboxylatreductase (proH), eine Glutamat-5-kinase (proJ), und eine Glutamat-5-semialdehyd-dehydrogenase (proA) kodieren. Mittels reverser Transkription von mRNA und anschließenden PCR-Analysen konnte gezeigt werden, dass die drei Gene ein Operon bilden. 10. Eine Quantifizierung der Transkriptmengen der Biosynthesegene proH, proJ und proA mittels quantitativer PCR in Zellen, die bei unterschiedlichen NaCl-Konzentrationen gezogen wurden, zeigte einen deutlichen Zusammenhang zwischen der Salinität des Mediums und der Menge an Transkript. Diese war umso höher, je höher die Salinität des Mediums war. Die maximale Transkriptmenge (6-fach) wurde bei einer Salzkonzentration von 2,5 M NaCl erreicht. Bei noch höherer Salzkonzentration sank die Transkriptmenge auf die ca. 5-fache Menge des Kontrollwertes ab. 11. Um die Regulation und Dynamik der Osmoregulation unabhängig vom Wachstum untersuchen zu können, wurde ein Zellsuspensions-System für H. halophilus etabliert, bei dem eine konzentrierte Zellsuspension direkt von geringen auf hohe Salzkonzentrationen überführt wurde und bei dem die Prozesse der Transkription, Translation und Solut-Biosynthese erhalten blieben. Beispielhaft wurde dieses System an der Produktion von Prolin nach einem Salzschock von 0,8 auf 2,0 M NaCl getestet. Es zeigte sich bei der Analyse, dass sich die Transkriptmengen unmittelbar nach dem Salzschock deutlich erhöhten und bereits nach 1,5 Stunden ein Maximum erreicht wurde. Verglichen mit dem Wert zu Beginn des Versuches waren die Transkriptmengen ca. 13-fach erhöht, sanken im weiteren Verlauf jedoch wieder ab und blieben bei einer 4-fachen Transkriptmenge konstant. Mit der Erhöhung der Transkriptmenge ging auch eine Erhöhung der Prolinkonzentration einher, die ein Maximum von ca. 6 μmol/mg Protein nach 6 Stunden erreichte. Auch diese Konzentration verringerte sich im weiteren Verlauf wieder und erreichte nach 20 Stunden den Ausgangswert. 12. Um den Einfluß diverser Anionen bzw. Osmolyte im Medium auf die Produktion von Prolin zu untersuchen, wurden Zellsuspensionen von H. halophilus einer Erhöhung der Osmolarität von 0,8 M auf 2,0 M unterzogen. Es zeigte sich dabei, dass die maximale Akkumulation von Prolin in Anwesenheit von Chlorid am höchsten war. Nitrat und Glutamat führten zu ähnlichen, aber leicht geringeren maximalen Konzentrationen (92 bzw. 83% des Chloridwertes). Gluconat führte noch zu einer Akkumulation von ca. 51%, während die anderen Osmolyte zu keiner Akkumulation führten. Eine Analyse der Transkriptmengen zeigte jedoch ein völlig anderes Bild. Während Chlorid, Nitrat und Gluconat zu vergleichbaren Anstiegen der Transkripmengen führten, war die maximale Transkriptmenge der Glutamatinkubierten Zellen 3-9 mal höher als in Vergleichszellen mit Chlorid. In anschließenden Titrationsexperimenten mit verschiedenen Glutamatkonzentrationen konnte gezeigt werden, dass eine minimale Konzentration von 0,2 M Glutamat ausreichend ist, um eine 90-fache Steigerung der Transkriptmenge herbeizuführen. 13. Als Antwort auf Hochsalz-Bedingungen akkumuliert H. halophilus neben Prolin auch Ectoin. Die Ectoinkonzentration bei 2,5 M NaCl war ca. 2-3 mal höher als in Zellen, die bei 1,0 M gezogen wurden. Die Bestimmung der intrazellulären Ectoin-Konzentrationen während des Wachstums zeigte außerdem, dass die Produktion von Ectoin wachstumsphasenabhängig ist. Die Konzentration in der stationären Phase war ca. 5-fach höher als in der exponentiellen Phase. Die Entwicklung der Ectoin- Konzentration verhielt sich somit reziprok zur Entwicklung der Prolin-Konzentration während des Wachstums. 14. Es wurde ein Cluster von drei Genen im Genom von H. halophilus identifiziert, deren Genprodukte die Biosynthese von Ectoin aus Aspartatsemialdehyd katalysieren. ectA kodiert dabei für eine putative Diaminobutyrat-Acetyltransferase, ectB für eine putative Diaminobutyrat-2-oxoglutarat-Transaminase und ectC für eine putative Ectoin-Synthase. Mittels reverser Transkription von mRNA und anschließenden PCR-Analysen konnte gezeigt werden, dass die drei Gene ein Operon bilden. 15. Die Transkription der ect-Gene war abhängig von der Salinität des Mediums. Ab 2,0 M stieg die Menge an RNA um das 10-fache an und erreichte bei 3,0 M ein Maximum mit der 23,5-fachen Menge. 16. Nach einem osmotischen Schock stieg die Konzentration an ect-mRNA signifikant und erreichte ein Maximum nach 3 - 4 Stunden. Das Maximum wurde somit 1,5 – 2,5 Stunden später erreicht als bei anderen Genen der Solute-Biosynthese wie etwa gdh1, das für eine Glutamatdehydrogenase, glnA2, das für eine Glutamin-Synthetase oder proH, das für eine Pyrrolin-5-Carboxylase kodiert. Die maximal erreichten Wert lagen 13-fach (ectA), 6,5-fach (ectB) und 3-fach (ectC) über dem Wert vor dem Salzschock. Gegen EctC wurden polyklonale Antikörper generiert. Western-Blot Analysen mit diesem Antikörper zeigten, dass die EctC-Menge nach 4 Stunden um das 2,5-fache stieg, dann aber wieder abfiel auf das 1,6 – 1,7-fache des Ausgangswertes. Der Rückgang an EctC fand keine Entsprechung in der gemessenen Ectoin-Konzentration, welche über einen Zeitraum von 18 Stunden kontinuierlich anstieg. Die maximale Konzentration nach 18 Stunden betrug das ca. 6,3-fache des Ausgangswertes. 17. Wurden H. halophilus Zellen mit anderen Osmolyten außer NaCl geschockt, so ergab sich folgendes Bild der Regulation der Ectoin-Biosynthese: (i) die Transkription der ect-Gene zeigte keine Chlorid-abhängige Regulation. Die maximale Transkriptmenge wurde in Gegenwart von Nitrat erreicht, wohingegen Gluconat zu vergleichbachen mRNA-Mengen führte wie Chlorid. Glutamat führte nur zu schwacher Stimulierung der Transkription. (ii) auf Ebene der Proteinmenge war zu sehen, dass die Menge an EctC nach osmotischem Schock vergleichbar war in Zellen, die mit Chlorid oder Nitrat inkubiert wurden. Gluconat führte nur zu einer 40%-igen Zunahme während andere Osmolyte nahezu wirkungslos auf die Menge an EctC blieben. (iii) die höchste Akkumulation an Ectoin nach einer plötzlichen Erhöhung der Osmolarität wurde erreicht mit Chlorid (6-fache Zunahme) gefolgt von Nitrat (5,6-fache Zunahme). Gluconat führte lediglich zu einer 3,3-fachen und Glutamat nur noch zu einer 2-fachen Steigerung der Ectoinkonzentration. Glutamat hat somit ähnliche Effekte wie Tartrat, Saccharose oder Sulfat. Succinat führte zu keiner Akkumulation und Glycin sogar zu einer deutlichen Abnahme. Die Produktion von Ectoin ist somit hauptsächlich abhängig vom Anion/Osmolyt und nur untergeordnet von der Osmolarität.
Quantum chromodynamics predicts the existence of a phase transition from hadronic to quark-gluon matter when temperature and pressure are sufficiently high. Colliding heavy nuclei at ultra-relativistic speeds allows to deposit large amounts of energy in a small volume of space, and is the only available experimental mean to produce the extreme conditions necessary to obtain the deconfined state. Numerous models and ideas were developed in the last decades to study heavy ion physics and understand the properties of extremely heated and compressed nuclear matter. With the ever increasing energy available in the center of mass frame (and thus number of particles produced) and the development of large acceptance detectors, it has become possible to study the fluctuations of physical quantities on an event-by-event basis, and access thermodynamical properties not present in particle spectra. The characteristics of the highly excited matter produced, e.g. thermalization, effect of resonance decay. . . can be investigated by fluctuation analyses. In fact, fluctuations are good indicators for a phase transition and a plethora of fluctuation probes have been proposed to pin down the existence and the properties of the QGP. We study various fluctuation quantities within the Ultra-relativistic Quantum Molecular Dynamics UrQMD and the quantum Molecular Dynamics qMD models. UrQMD is based on hadron and string degrees of freedom and allows to disentangle purely hadronic effects. In contrast, the qMD model includes an explicit transition from quark to hadronic matter and can serve to test adequate probes of the initial QGP state. We show that the qMD model can reasonably reproduce various experimental particles rapidity distributions and transverse mass spectra in wide energy range. Within the frame of the dynamical recombination procedure used in qMD, we study the enhancement of protons over pions (p/π) ratio in the intermediate pt range (1.5 < pt < 2.5). We show that qMD can reproduce the large p/π ≈ 1 observed experimentally at RHIC energies at hadronization. However, the subsequent decay of resonances makes the ratio fall to values incompatible with experimental data. We thus conclude that resonance decay might have a drastic influence on this observable in the quark recombination picture. Charged particles multiplicity fluctuations measured at SPS by the NA49 collaboration are enhanced in midperipheral events for Pb+Pb collisions at Elab = 160 AGeV. This feature is not reproduce by hadron-string transport approaches, which show a flat centrality dependence, within the proper experimental acceptance and with the proper centrality selection procedure. However, we show that the behavior of multiplicity fluctuations in transport codes is similar to the experimental result in full 4π acceptance. We identify the centrality selection procedure as the reason for the enhanced particle multiplicity fluctuations in midperipheral reactions and argue that it can be used to distinguish between different scenarios of particle productions. We show that experimental data might indicate a strong mixing of projectile and target related production sources. Strangeness over entropy K/π and baryon number over entropy p/π ratio fluctuations have been measured by the NA49 experiment in the SPS energy range, from Elab = 20 AGeV up to Elab = 160 AGeV. We investigate the sensitivity of this observable to kinematical cuts and discuss the influence of resonance decay. We find the dynamical p/π ratio fluctuations to increase with beam energy, in agreement with the measured data points. On the contrary, the dynamical K/π ratio fluctuations are essential flat as a function of centrality and depend only weakly on the kinematical cuts applied. Our results are in line with the simulations performed earlier by the NA49 collaboration in their detector acceptance filter. Finally, we focus on the correlations and fluctuations of conserved charges. It was proposed that these fluctuations are sensitive to the fractional charge carried by the quarks in the initial QGP stage and survive the whole course of heavy ion reactions. A crucial point is the influence of hadronization that may relax the initial QGP fluctuation/correlation signals to their hadronic values. We use the quark Molecular Dynamics qMD model to disentangle the effect of recombination-hadronization on charged particles ratio fluctuations, charge transfer fluctuations, baryon number-strangeness correlation coefficient and various ratios of susceptibilities (i.e. correlations over fluctuations). We find that the dynamical recombination procedure implemented in the qMD model destroys all studied initial QGP fluctuations and correlations and might ex- plain why no signal of a phase transition based on event-by-event fluctuations was found in the experimental data until now.
The chemiosmotic theory suggested by Peter Mitchell (Mitchell, 1961, Nature 191:144-148; see Mitchell, 1979, Science 206:1148-1159 for review) postulated that the energy released upon the oxidation of electron donor substrates is transiently stored as electrochemical proton potential, delta-p across energy-transducing membranes, which acts then as the driving force for the ATP synthesis. Membrane protein complexes can both generate and utilise a transmembrane electrochemical proton potential, either by transmembrane proton transfer or by transmembrane electron transfer coupled to protolytic reactions on opposite sides of the membrane. The dihaem-containing membrane protein complex quinol:fumarate reductase (QFR) from the anaerobic epsilon-proteobacterium Wolinella succinogenes apparently combines both of these mechanisms (Haas et al, 2005, Biochemistry 44:13949-13961; Lancaster et al, 2005, PNAS 102:18860–18865; Mileni et al, 2005, Biochemistry 44:16718-16728; Madej et al, 2006, EMBO J 25:4963-4970). QFR is the terminal enzyme of anaerobic fumarate respiration that allows bacteria to use fumarate as the terminal electron acceptor (Kröger, 1978, Biochim Biophys Acta 505:129-45; Lancaster, 2004, In: Respiration in Archaea and Bacteria Volume 1:57-85). QFR couples the two-electron reduction of fumarate to succinate to the two-electron oxidation of quinol to quinone. QFR contains two haem b groups bound by the transmembrane subunit C, which are termed the ‘proximal haem’, bP, and the ‘distal haem’, bD, according to the relative proximity to the hydrophilic subunits A and B (Lancaster et al, 1999, Nature 402:377-85). The two-electron transfer via the two haem groups has been proposed (Lancaster, 2002, Biochimica et Biophysica Acta 1565:215-231) and demonstrated (Madej et al, 2006, EMBO J 25:4963-4970) to be coupled to a compensatory, parallel transfer of two protons via a transmembrane proton transfer pathway. The two most prominent constituents of the proposed pathway were suggested to be the haem bD ring C propionate and the side chain of amino-acid residue Glu C180, after which the proton transfer pathway was named the ‘E-pathway’ (Lancaster, 2002, Biochimica et Biophysica Acta 565:215-231). The essential role of Glu C180 was supported by site-directed mutagenesis and structural and functional characterization of the enzyme E180Q, where the Glu C180 was replaced with a Gln residue (Lancaster et al, 2005, PNAS 102:18860–18865). Moreover, multiconformer continuum electrostatics (MCCE) calculations (Haas and Lancaster 2004, Biophys J 87:4298-4315) and Fouriertransformed infrared (FTIR) spectroscopy experiments (Haas et al, 2005, Biochemistry 44:13949-13961) indicated the Glu C180 side chain to undergo a combination of a conformational change and protonation upon haem reduction. The contribution of haem bD propionate is less clear, however, a combination of 13C labelling of the haem propionates with redox-induced FTIR experiments (Mileni et al, 2005, Biochemistry 44:16718-16728) and MCCE calculations (Haas and Lancaster, 2004, Biophys J 87:4298-4315) support a change in protonation, possibly accompanied by a change in environment upon haem reduction. These experiments and their results strongly support the existence of the ‘E-pathway’ which is transiently open during the reduction of the haem groups and blocked in the oxidized state of the enzyme (Lancaster, 2002b, Biochim Biophys Acta 1565:215-231). All available crystal structures of the QFR, however, are those of the oxidized enzyme. Therefore, it is advantageous to perform simulations of various redox states of the enzyme to determine for instance, how the side-chain of Glu C180 and haem bD ring C propionate behave upon changes of the redox states of the haem groups and why is the ‘E-pathway’ blocked in the oxidized state of the enzyme. Although the distal haem ring C propionate and Glu C180 were identified as the most prominent components of the proton transfer pathway, it was not clear, on the basis of the structure, how proton transfer could occur between them. In addition, two constituents are not enough to span the membrane region and the additional participants in the proton transfer pathway must be identified. Since an atomistic investigation of proton transfer in this system is not yet possible experimentally, I used available theoretical methods such as classical molecular dynamics (MD) simulation (Alder and Wainwright, 1959, J Phys Chem 31:459-466; McCammon et al, 1977, Nature 267:585-590) and Q-HOP molecular dynamics (Q-HOP MD) simulation (Lill and Helms, 2001, J Chem Phys 115:7993-8005) to investigate the postulated mechanism of electron coupled proton transfer in QFR. MD simulations allowed us to move away from static difference pictures obtained from FTIR experiments and MCCE calculations. The advantage of the MD simulations over the experiments and the simulations performed so far is that the time-dependent properties could now be analyzed. The behaviour of various residues and their side-chains and any environmental changes may be directly observed during MD simulations. Although classical MD simulations cannot be used to study proton transfer reactions, they can provide information on formation of configurations that would allow either direct proton transfer between donor and acceptor residues or indirect proton transfer mediated by water molecules. To avoid the static protonation of residues which is inherent in classical MD simulations, Q-HOP MD simulations were performed which explicitly describe proton transfer reactions by allowing the change of the protonation state of residues ‘on the fly’. The structures obtained after classical molecular dynamics simulations ....
The thesis is devoted to the study of the Antarctic polar vortex, mainly by analyzing data collected during APE-GAIA (1999) and ASHOE (1994) campaigns and recorded by the ADEOS satellite (1996-1997), and to improvement of the chromato-graphic processing schemes. A general introduction and overview of the campaigns and instruments relevant to the present work are given in Chapters 1 and 2. A relatively large part of the thesis (Chapters 3-5) is on improvement of the analysis of raw chromatographic data recorded during in-flight measurements of the trace gases. A Gaussian non-straight-base-line method, i.e. the Gaussian processing scheme (Chapter 3), is developed for better evaluation of the chromatographic peak size. Furthermore, a statistical cross-correlation method (Chapter 5) based on statistical behaviour of the whole chromatogram series fNchrg recorded, e.g., during a research flight or laboratory calibration, is developed and applied to measure the low-concentration trace gases. As demonstrated for HAGAR's chromatograms (HAGAR - High Altitude Gas Analyzer), the combination of the Gaussian fitting scheme for individual chromatograms and the statistical cross-correlation method for a series of subsequent chromatograms considerably improves and stabilizes quantitative analysis of in-flight chromatographic data. In this case, the detection accuracy of weak and noisy chromatographic signals can be improved by up to 40 %. A particular attention is paid to the in-flight two-standard calibration method. For this method, a special procedure, that allows to evaluate and effectively remove a weak background chromatographic signal associated with residual molecules in the carrier gas N2, is proposed and coded (Chapter 4). The developed approaches and methods are completely automized and, therefore, can be used for processing of in-flight chromatograms of recent and future field campaigns. The main part of the thesis (Chapters 6-8) deals with a two-dimensional quasi-Lagrangian coordinate system ... , based on a long-lived stratospheric trace gas i, and its systematic use for i = N2O in order to describe the structure of a well-developed Antarctic polar vortex, linearization and compactization of the tracer-tracer correlations in the polar vortex core (i.e. the stratospheric dynamics in this area), and the differential ozone losses in the Antarctic polar vortex area. In the coordinate system ... (...-method, Chapter 6), which refers to a well-developed polar vortex, the mixing ratio Âi is the vertical coordinate and ... = .... i is the reference profile in the vortex core) is the meridional coordinate. The quasi-Lagrangian coordinates ... are much more long-lived comparing with the standard quasi-isentropic coordinates, potential temperature ... and equivalent latitude ..e, do not require explicit reference to geographic space, and therefore well-suited for studying the dynamics of the Antarctic polar vortex and the relevant ozone loss processes. By using the introduced coordinate system ... to analyze the well-developed Antarctic vortex investigated in the APE-GAIA campaign, it is shown, in concurrence with the conclusion of A. M. Lee et al. (2001), that the Antarctic vortex area can be described in terms of the well-mixed and well-isolated vortex core, relatively wide vortex boundary region and adjoining surf zone. In this case, the reference profile ... i , which is compact in a well-developed and isolated polar vortex core [J. B. Greenblatt et al. (2002)], can be found by combining airborne (and/or balloon) data with high-altitude satellite measurements. A criterion, which uses the local in-situ measurements of Âi = Âi(£) and attributes the inner vortex edge to a rapid change (±-step) in the meridional pro¯le of the mixing ratio..., is developed in Chapter 6 to determine the (Antarctic) inner vortex edge. In turn, the outer vortex edge of a well-developed Antarctic vortex is proposed to attribute to the position of a local maximum of ...H2O in the polar vortex area. For a well-developed Antarctic vortex, the ...-parametrization of tracer-tracer correlations allows to distinguish the tracer-tracer inter-relationships in the vortex core, vortex boundary region and surf zone (Chapter 7). This is clearly illustrated by analyzing the tracer-tracer relationships Âi ¡ ÂN2O obtained from the in-situ data of the APE-GAIA campaign for i = CFCl3 (CFC-11), CF2Cl2 (CFC-12), CBrClF2 (H-1211) and SF6. The solitary anomalous points in the ...CFC11 ¡ ÂN2O correlation, observed in the Antarctic vortex core during the APE-GAIA and ASHOE campaigns, are interpreted in terms of small-scale localized differential descent. As detailed in Chapter 8, the quasi-Lagrangian coordinate system fÂN2O; ¢ÂN2Og is an effective tool for evaluation of the differential ozone losses in the polar vortex area. With this purpose, a two-parametric reference function ...O3 = F(...), which characterizes the unperturbed O3 distribution in the early winter polar vortex area, is introduced to separate and quantify in terms of the meridional coordinate ...2O the differential ozone losses in the vortex core and vortex boundary region. The method is applied to analyze the ozone depletion in the Antarctic stratosphere during the austral spring 1999 (APE-GAIA campaign). In Chapter 9, the main results of the thesis are summarized.
The ABC protein ABCE1, also called HP68 or RNase L inhibitor (RLI), is one of the most conserved proteins in evolution. It is universally expressed in eukaryotes and archaea, where ABCE1 is essential for life. ABCE1 plays a crucial role in translation initiation and ribosome biogenesis, however, the molecular mechanism of ABCE1 remains unclear. In addition to two ABC ATPase domains, ABCE1 contains a unique N-terminal region with eight conserved cysteines predicted to coordinate iron-sulfur (Fe-S) clusters. To analyze the function of ABCE1, the hyperthermophilic crenarchaeote Sulfolobus solfataricus was chosen as a model system. S. solfataricus ABCE1 was overexpressed homologously in S. solfataricus and heterologously in E. coli. Noteworthy, for tagged-protein production in S. solfataricus a novel expression system based on a virus shuttle vector was established. This is the first example for a successful overexpression and purification of isolated full-length ABCE1. For the first time it was shown that ABCE1 indeed bears biochemical properties of an ABC protein even though it has unique features. Remarkably, the nucleotide binding domains (NBDs) of ABCE1 bound ATP and AMP, but were functionally non-equivalent in ATP hydrolysis. Mutations of conserved residues in the second NBD led to a hyperactive ATPase, which implies an intramolecular mechanism of dimer formation. Truncation of the Fe-S cluster domains did not influence ATPase activity. The Fe-S clusters of ABCE1 were analyzed by biophysical and biochemical methods. As presented in this study, ABCE1 harbors two essential diamagnetic [4Fe-4S]2+ clusters, one ferredoxin-like cluster formed by cysteines at position 4/5/6/7 and one unique ABCE1 cluster formed by cysteines at position 1/2/3/8. ABCE1 was found to be associated with RNA after purification from S. solfataricus and bound ribosomal RNA in vitro. In addition, ABCE1 showed homo-oligomerization and appeared to form a hexameric complex of ~440 kDa, which was RNase sensitive. Archaeal ABCE1 associated with ribosomes, however, the unique Fe-S clusters of ABCE1 were not required for this interaction. Although archaeal ABCE1 assembled with ribosomes and ribosomal RNA, ABCE1 proved not to be essential for translation in S. solfataricus and did not interact with archaeal initiation factors. Nevertheless, the ABCE1 gene is one of the few genes conserved between archaea and eukaryotes and fulfills a universal task, which needs further characterization.
21 Hsfs belonging to classes A, B and C were identified in Arabidopsis following the sequencing of its genome. 1.) Cloning of full length and CTD chimeric constructs followed by transient reporter assays in tobacco protoplast using GUS fusion constructs of the promoters of Hsp17.4-CI, synthetic (HSE9) and APX2 showed Hsfs A1a, A1b, A1d, A1e, A2, A3 and A9 to be active. CTDs of Hsfs A7a, A7b and HsfC1 had activity but they showed poor DNA binding in reporter assays. Hsfs A1a, A1b, A1d, A1e, A2 and A3 were able to induce the expression of endogenous Hsps in tomato protoplasts. Interesting differences in promoter selectivity were observed for several Hsfs. 2.) RT-PCR and microarray analysis showed the Hsfs to be differentially expressed depending on tissue, abiotic and biotic stress, hormone and developmental s ge. Interesting patterns of coexpressed Hsfs were observed under different stresses and developmental stages. 3.) HsfA1b was found to be active on the plasmid borne PHsf:GUS reporters of Hsfs A1d, A2, A4a, A7b and B4 when tested in tobacco mesophyll protoplasts. Hsfs A1d, A2, A4a, A7b and B4 when tested in tobacco mesophyll protplasts. HsfA2 was inactive on PHsfA:GUS. HsfB1 showed repression of endogenous activity on several PHsf:GUS reporter constructs. 4.) The transcriptional regulation under heat stress and promoter organization of HsfA2 and FtSH4 (a metalloprotease gene oriented in a head to head fashion with HsfA2 in the Arabidopsis genome, sharing a common promoter region) was studied. The transcripts of FtSH4 and HsfA2 coaccumulated under heat stress. HsfA1b was active on PHsfA2:GUS and PFtSH4:GUS. Hsf binding sites on the intergenic region were determined using promoter deletion constructs in tobacco and Arabidopsis protoplasts. A bidirectional regulation of HsfA2 and FtSH4 by HsfA1b was observed in tobacco protoplast. 5.) Microarray analysis of a HsfA2 T-DNA insertion line vs. wild type Col-0 under heat stress conditions led to identification of a subset of target genes to be severely affected in the absence of HsfA2. Apart from several Hsps (heat stressproteins) and APX2 (Ascorbate peroxidase 2, oxidative stress scavenger), several other unknown genes are affected. APX2 was the most severely affected among them. HsfA2 was able to induce the transcription from its target gene promoters in fusion to GUS in transient reporter assays in tobacco protoplast. The HSE cluster to which HsfA2 binds on the APX2 promoter was also mapped by the same technique. The direct binding of HsfA2 to the promoter of selected target genes in the Arabidopsis genome was also demonstrated by chromatin immunoprecipitation studies.
Two distinct mechanisms contribute to the development of blood vessels: vasculogenesis, which is the de novo formation of vascular structures from progenitor cells, and angiogenesis, the formation of new blood vessels from pre-existing ones.
Angiogenesis is a highly ordered and carefully regulated multi-step process, during which the precise spatio-temporal interaction between endothelial and mural cells, i.e. smooth muscle cells and pericytes, is prerequisite for the formation of a functional blood vessel. The crosstalk between these two latter cell ty pes is mediated indirectly by various
secreted growth factors, and directly through cell-cell and cell-matrix interactions. The secretory epidermal growth factor-like protein 7 (EGFL7) has been implicated to
play an important role in the regulation of smooth muscle and endothelial cell recruitment and vascular tube formation. However, in-depth investigation of the underlying molecular mechanism has so far been hampered by the lack of functional recombinant EGFL7. In this study for the first time full length EGFL7 was successfully expressed as a His 6- tagged fusion protein from insect cells using the Baculovirus expression vector system. Recombinant EGFL7 was purified in a two-step protocol involving ion metal affinity chromatography and gel filtration. Furthermore, recombinant EGFL7 was
purified from human embryonic kidney EBN A 293 cells using a similar approach, allowing the production of high amounts of recombinant EGFL7 protein in its native state, with proper post-translational processing and full biological activity. Detailed analysis of the post-translational processing of recombinant EGFL7 and EGFL7-mutants revealed extensive proteolytic processing by protein convertases both at the N- and the C-terminus, the latter being prerequisite for EGFL7 secretion. Furthermore, secreted EGFL7 protein was shown to bind to the extracellular matrix and the responsible heparin-binding domain of EGFL7 was mapped to its N-terminal
portion. Purified recombinant EGFL7 protein was tested for its functionality using cell migration assays, cell proliferation studies and in vivo matrigel studies in mice. In the
modified Boyden chamber migration assay, recombinant EGFL7 proteins inhibited PDGF-BB-induced smooth muscle cell migration. Moreover, recombinant EGLF7 proteins strongly inhibited PDGF-BB-induced proliferation of smooth muscle cells, while it did not affect VEGF induced proliferation of endothelial cells. When applied in the in vivo matrigel plug assay, EGFL7 proteins induced a strong pro-angiogenic response, comparable with that of VEGF on an equimolar basis. Moreover, EGFL7 expression was strongly induced in endothelial cells in response to VEGF stimulation. These novel findings demonstrate the important function of EGFL7 in angiogenesis and are well in line with previous results. They demonstrate a cell specific action of EGFL7 on the different cell types involved in vessel formation, which is a prerequisite for a regulatory function in cell-to-cell crosstalk. Based on the results described here, the following model can be proposed: VEGF, a known strong initiator of angiogenesis, induces endothelial cell proliferation and migration, allowing the
escape from the comparatively rigid structure of a functional vessel to form an angiogenic sprout. At the same time VEGF induces the expression of EGFL7 in endothelial cells. EGFL7 is expressed, proc essed and secreted from these cells. While EGFL7 has no known effect on endothelial cells, it inhibits smooth muscle cell proliferation and migration, providing a mechanism to prevent pre-mature stabilization of the forming vessel. The availability of purified recombinant EGFL7 will be helpful in the detailed characterization of the underlying molecular mechanism of EGFL7 action, including the identification of the putative EGFL7 receptor, and will allow - together with knock-out experiments in mice - the exploration of the additional biological functions of EGFL7. Moreover, considering the strong pro-angiogenic effect of EGFL7 in vivo, it would be also of a great therapeutic interest to investigate its role in the development of tumor vasculature. The insights into these molecular mechanisms might provide a novel approach for the development of anti tumor therapies.
Analysis of coding principles in the olfactory system and their application in cheminformatics
(2007)
Unser Geruchssinn vermittelt uns die Wahrnehmung der chemischen Welt. Im Laufe der Evolution haben sich in unserem olfaktorischen System Mechanismen entwickelt, die wahrscheinlich optimal auf die Erfüllung dieser Aufgabe angepasst sind. Die Analyse dieser Verarbeitungsstrategien verspricht Einblicke in effiziente Algorithmen für die Kodierung und Verarbeitung chemischer Information, deren Entwicklung und Anwendung dem Kern der Chemieinformatik entspricht. In dieser Arbeit nähern wir uns der Entschlüsselung dieser Mechanismen durch die rechnerische Modellierung von funktionellen Einheiten des olfaktorischen Systems. Hierbei verfolgten wir einen interdisziplinären Ansatz, der die Gebiete der Chemie, der Neurobiologie und des maschinellen Lernens mit einbezieht.
Many tax-codes around the world allow for special taxable treatment of savings in retirement accounts. In particular, profits in retirement accounts are usually tax exempt which allow investors to increase an asset's return by holding it in such a retirement account. While the existing literature on asset location shows that risk-free bonds are usually the preferred asset to hold in a retirement account, we explain how the tax exemption of profits in retirement accounts affects private investors' asset allocation. We show that total final wealth can be decomposed into what the investor would have earned in a taxable account and what is due to the tax exemption of profits in the retirement account. The tax exemption of profits can thus be considered a tax-gift which is similar to an implicit bond holding. As this tax-gift's impact on total final wealth decreases over time, so does the investor's equity exposure. JEL Classification Codes: G11, H24
Cytochrome b561 (cyt b561) proteins are members of the recently identified eukaryotic ascorbate reducible protein family named CYBASC (CYtochrome B, ASCorbate reducible). CYBASC proteins are di-heme-b-containing membrane proteins that catalyze the transmembrane electron transfer from ascorbate. The function of the CYBASC proteins has been correlated with ascorbate recycling and/or iron facilitation uptake. Therefore, investigations on this family are of great interest as ascorbate is one of the most powerful antioxidants and iron is essential for cell survival both in animals and plants. As the amino acid sequence conservation of animal and plant CYBASC proteins is relatively high, all CYBASC members are proposed to share the same structural motifs. However, no three-dimensional structure of any representative member of the CYBASC family has been determined to date. In the Arabidopsis thaliana (A. thaliana) genome, two complete putative CYBASC open reading frames (ORFs), artb561-a and artb561-b were identified. In this thesis, these two A. thaliana CYBASC ORFs, encoding for Acytb561-A and Acytb561-B proteins respectively, were investigated and obtained main results are listed. 1. A. thaliana CYBASC proteins were heterologously produced in Pichia pastoris and Escherichia coli and purified by a single-step immobilized metal affinity chromatography (IMAC). To facilitate detection and purification, the recombinant A. thaliana CYBASC proteins were produced in both expression systems with the histidine affinity tag. Pure and stable preparations of the cytochromes were obtained via a single-step IMAC in sufficient amounts to perform biochemical characterizations. 2. Detergent solubilized recombinant Acytb561-A and Acytb561-B are dimers. As previously suggested for other CYBASC proteins, analytical gel filtration experiment suggested that both detergent solubilized cytochromes are dimers. 3. Spectroscopic features of Acytb561-B differed from those of previously described bovine chromaffin granule cyt b561. A distinctive feature of the first identified CYBASC protein, the cyt b561 from bovine chromaffin vesicles of adrenal medulla (Bcytb561-CG), is that its differential visible absorbance spectra (visible-spectra) revealed an asymmetric α-band with a maximum at 562 nm and a clear shoulder at 557 nm. This feature was recently used to discriminate CYBASC proteins from not-CYBASC proteins. However, in this thesis, it is shown for the first time that not all CYBASC proteins display in their reduced-minus-oxidized visible-spectra an asymmetric α- band and therefore, this feature can not be used as a discriminating CYBASC characteristic. 4. Ascorbate dependent reduction of the A. thaliana CYBASC proteins is inhibited by diethylpyrocarbonate (DEPC). As previously reported for the Bcytb561-CG, the ascorbatedependent reduction of the A. thaliana CYBASC proteins was inhibited by DEPC treatment. In addition, the ‘ascorbate protectant’ effect against DEPC that was observed on the Bcytb561-CG was also observed on the Acytb561-A and Acytb561-B proteins. Furthermore, as the physiological electron donor of all CYBASC proteins is supposed to be ascorbate, ascorbate-affinity of Acytb561- A and Acytb561-B was monitored and was found to be in the same range of the one of the Bcytb561- CG. 5. A. thaliana CYBASC proteins are Fe3+-chelate reductases. Recently, the Fe3+-chelate reductase activity of various CYBASC proteins was presented. In this thesis, it is shown that also both A. thaliana CYBASC proteins reduced Fe3+-chelates such as Fe3+-EDTA and Fe3+-citrate. Consistently, heme potentiometric reductive-oxidative titration of purified Acytb561-A and Acytb561-B indicated that the midpoint potential of the two heme centres of both cytochromes was lower than the one of those Fe3+-chelates. The values of both heme centre potentials of Acytb561-A and Acytb561-B are also consistent with the observation that both cytochromes were only partially reducible by ascorbate and were fully reduced with the non-physiological reductant Na-dithionite. In summary, this work describes the heterologous production, purification and initial characterizations of two distinct CYBASC proteins from A. thaliana: Acytb561-A and Acytb561-B. Biochemical characterization of these cytochromes showed that the shape of the α-band in the differential spectra is not a discriminating factor for CYBASC proteins but it is likely the DEPC sensitivity and the Fe3+-chelate reductase activity. Establishment of a purification strategy to obtain sufficient amounts of monodispersed and stable A. thaliana CYBASC proteins has also enabled initial screening of three dimensional crystallization conditions which are a prerequisite for a deeper understanding of this new eukaryotic redox enzyme family.
Background: False aneurysms at the puncture site develop in up to 8 % after catheter procedures. They can be treated surgically or by ultrasound guided manual compression. A new method is to inject thrombin into the aneurysm under ultrasound guidance. We evaluated safety and efficacy of this approach in a multicenter registry. Methods: In 595 consecutive patients (age: 31-94 years, median 70) a pseudoaneurysm (593 femoral arteries, 2 brachial arteries) was diagnosed 0 to 250 days (median 3 days) after a catheter procedure. The diameter of the aneurysm ranged from 0.5 x 0.5 x 0.5 (L x W x D) to 8x11x16 cm (median 2 x 2 x1.6 cm). 20 U to 4000 U of thrombin solution (median 400 U) were injected percutaneously into the aneurysm under ultrasound guidance. Results: The procedure was technically successful in 587/595 (99%) patients. The aneurysms were thrombosed after the first injection in 531 patients (89 %). Thirty-eight (6%) patients needed a second injection and 8 (1%) patients a third injection because residual flow in the aneurysm was visible at follow-up. In 4 additional patients (0.7%) the thrombosis of the aneurysms was delayed and occurred only after 24 hours to 7 days. 6 (1%) patients surgery was performed after successful closure of the aneurysm to remove the resulting haematoma. The overall technical success rate was 99% (587/595) and clinical success was achieved in 572/595 (96%) patients. Eight (1%) other patients underwent surgery due to thrombin injection failure. Complications occurred in 9 patients (1.5%): Intravascular thrombus formation (n=3), deep venous thrombosis (n=3), pulmonary embolism due to deep venous thrombosis (n=1), transient paresthesia in the leg during injection (n=3). Conclusion: Ultrasound guided thrombin injection is a safe, painless, effective and rapid alternative to treat false aneurysms. Complications and recurrent pseudoaneurysms are very rare. It has become the treatment of choice in our institution.
Deferred imitations assess declarative memory in infants. Many cross-sectional and a few longitudinal studies revealed that, with development, infants learn faster,and retain more target actions over longer retention intervals. Longitudinal stabilities are modest and increase through the second year. To date, there are only few multivariate deferred imitation studies pointing to interactions between declarative memory, language and self-development. However, as these studies applied variable-centered data analysis approaches, the individual stance was not taken into account.Therefore, the present dissertation focuses on the explanation of inter-individual differences of deferred imitation through the second year. In the multivariate, longitudinal Frankfurt Memory Study (FRAMES), declarative memory (deferred imitation), non-declarative memory (train task), as well as cognitive, language, motor, social, emotional and body self-awareness development (Developmental Test for 6-month- to 6-year-olds, ET6-6) were assessed on three measurement occasions (12-, 18- and 24-month-olds). From a psychometric perspective, sound tests for the assessment of deferred imitation in the respective age groups were developed (Paper 1 & 2). Reliability analyses (Paper 3) indicated relatively high short-term-stability for the deferred imitation test (12-month-olds). The co-development of declarative and nondeclarative memory in 12- and 18-month-olds provided evidence for discriminative validity (Paper 4). Longitudinally, deferred imitation performance tremendously increased throughout the second year, and performance was moderately stable between 12 and 18 months and stability increased between 18 and 24 months. Using a person-centered analysis approach (relative difference scores; cluster analysis), developmental subgroups were extracted out of the total sample. These groups differed in terms of mean growth and stability. However, between the first and second measurement occasion, the groups did not differ with respect to motor, cognitive and language development (Paper 5). Using the data of three measurement occasions, subgroups were extracted showing significant differences with respect to language, motor and body self-awareness development (Paper 6). The results are discussed against the background of infancy development theories.
Both practitioners and academics agree about the importance of price and its direct influenceon consumers’ purchase decision as well as the company profit. In the reality, we rarely see a
single price for a given product. One visit in a store already shows that consumers face many various prices. This strategy of differential prices allows to increase profit but also improves consumers’ situation and increases welfare. A wide range of various price differentiation mechanisms exists on the market which makes price differentiation a very interesting phenomenon. Additionally, market developments constantly allow for new price differentiation applications. In this work, I research a fascinating topic of price differentiation, its various forms
and new application possibilities in changing market areas.
I investigate some of the inert phases in three-flavor, spin-zero color-superconducting quark matter: the CFL phase (the analogue of the B phase in superfluid 3He), the A and A* phases, and the 2SC and sSC phases. I compute the pressure of these phases with and without the neutrality condition. Without the neutrality condition, after the CFL phase the sSC phase is the dominant phase. However, including the neutrality condition, the CFL phase is again the energetically favored phase except for a small region of intermediate densities where the 2SC/A* phase is favored. It is shown that the 2SC phase is identical to the A* phase up to a color rotation. In addition, I calculate the self-energies and the spectral densities of longitudinal and transverse gluons at zero temperature in color-superconducting quark matter in the CFL phase. I find a collective excitation, a plasmon, at energies smaller than two times the gap parameter and momenta smaller than about eight times the gap. The dispersion relation of this mode exhibits a minimum at some nonzero value of momentum, indicating a van Hove singularity.
Transport of proteins into or across cellular membranes is mediated by the conserved and ubiquitous Sec-machinery. The Sec-homologue in the inner membrane of Escherichia coli is SecYEG. Sec-mediated insertion of numerous membrane proteins is aided by YidC, another protein integral to the inner membrane of Escherichia coli. YidC fulfils in addition the integration of a variety of membrane proteins Sec-independently. It belongs to a conserved but structurally uncharacterised family of proteins important for membrane protein biogenesis and comprises homologues in mitochondria and chloroplasts. By modification of a former crystallisation protocol two-dimensional crystals of SecYEG were grown in presence of the signal sequence peptide of LamB. Recording of structural data by electron cryo-microscopy and calculation of a difference structure comparing a former SecYEG projection structure with the one of SecYEG crystallised in presence of the substrate revealed several new and vacant densities. These hint to signal peptide binding close to the translocation pore and to significant rearrangements in proximity to the lateral exit site for transmembrane domains in SecYEG. The difference structure suggests that dimeric SecYEG is an asymmetric molecule consisting of one active and one inactive SecYEG monomer. Detergent removal from a mixture of purified YidC and lipids produced two-dimensional crystals that were highly dependent on the ionic strength and lipid composition for their growth. Electron cryo-microscopy on the frozen-hydrated crystals and image processing visualised structural details at about 10 Å resolution. Averaging two alternative projection structures in p2 and p121_a symmetry, respectively, yielded essentially the same features. Four YidC monomers form one unit cell (dimensions 82 x 71 Å, included angle 85 ° and 90 °, respectively) and seem to be arranged as two sets of dimers integrated in an anti-parallel fashion into the membrane. An area of low density in the centre of each YidC monomer resembles possibly a constriction of the membrane, which could have particular relevance for the integration of substrate proteins into the lipid bilayer.
This work analyses several granitic bodies of the Variscan Orogen of Central and Western Europe in order to improve our knowledge about different aspects of their evolution, regarding their ascent and emplacement mechanisms, as well as their deformation history. In the Iberian Massif two granitoid bodies, namely the La Bazana pluton and the Nisa-Alburquerque batholith, were studied in order to decipher their ascent and emplacement history. The La Bazana pluton is a small, sub-circular body in map view that intruded into rocks of the Ossa-Morena Zone in the core of a late upright antiform. Its three-dimensional drop-pipe shape, its internal dome foliation pattern and the structure of the host rock suggest that the magma ascended and emplaced diapirically. The Nisa-Alburquerque batholith is a large body that intruded into rocks of the Central Iberian Zone, the Central Unit, and the Ossa-Morena Zone. Its cartographic shape is elongate and parallel to the NW—SE to WNW—ESE Variscan structures. In the light of the available structural data and the gravimetric models, the intrusion is viewed as a continuous lateral magma flow from the eastern root guided towards the west through the southern limb of a kilometre-scale antiform. As mass-transfer mechanisms, a combination of rigid translation of the country rocks, stoping, and possibly ballooning is proposed. In the Bohemian Massif several small granitoid bodies showing a strong solid-state deformation were studied in order to integrate their tectonometamorphic history in the geotectonic framework of the south-western Bohemian Massif, focusing principally on the deformation phase referred to as D3. Four ductile deformation phases are proposed for the study area. D1 produced high-temperature fabrics under upper amphibolite to granulite facies conditions. Its kinematics is unknown. D2 occurred under amphibolite to upper greenschist facies conditions under N—S to NNW—SSE compression. It is responsible for a subvertical NW—SE striking foliation in migmatites developed under dextral simple shear and for the deformation at the Bayerischer Pfahl shear-zone system at its earlier stages. Many granitoid dykes and stocks were found to be affected by sinistral shear along subvertical planes trending ENE to ESE. Since this deformation, which is called D3 in the present work, is not compatible with a N—S to NNW—SSE compression, it is proposed that these sinistral shear zones in granites do not belong to the Bayerischer Pfahl shear-zone system and constitute themselves a separated one, which is called “D3 shear-zone system”. D3 took place under upper greenschist to lower amphibolite facies conditions (~480-550°C). Both the intrusion and the deformation of the granites affected by D3 occurred at deep to intermediate levels of the crust, whereas the deformation took place under NE—SW compression. Datings on two of the deformed granites yielded 324.4 ± 0.8 Ma and 315.0 ± 1.0 Ma: Thus, the age of D3 is most probably ~315 Ma. The intrusion of most of the sheared granitoids was pre-kinematic with respect to D3. After D3 the N—S to NNW—SSE compression which governed D2 was restored, giving way to the next deformation phase D4, which was linked to further deformation at and next to the principal shears of the Bayerischer Pfahl shear-zone system under greenschist facies conditions. The causes for the change of the stress field leading to a NE—SW compression during D3 might be related to (1) global changes in the dynamics of the tectonic plates in late Variscan times, (2) orogenic collapse leading to the sinking of the Teplá-Barrandian and lateral extrusion of the surrounding Moldanubian rocks, (3) distortion of the regional stress field by local intrusion of large stocks, such as the Saldenburg granite of the Fürstenstein Massif, or (4) distortion of the regional stress field due to the existence of ephemeral releasing bends in the Bayerischer Pfahl shear zone during its early evolution.
A detailed understanding of how potassium channels function is crucial e. g. for the development of drugs, which could lead to novel therapeutic concepts for diseases ranging from diabetes to cardiac abnormalities. An improved understanding of channel structure may allow researchers to design medication that can restore proper function of these channels. This is particularly important for KCNQ channels, since four out of five family members are involved in human inherited disease. In addition to structure and function relationships the determinants which govern assembly of KCNQ subunits are decisive to understand the physiological role of the KCNQ channel family members. Many details of KCNQ channel assembly remain incompletely understood. Previous work has shown that the subunit-specific heteromerisation between KCNQ subunits is determined by a ~115 amino acid-long subunit interaction domain (si) within the C-terminus (Schwake et al., 2003). Recently, Jenke et al. (2003) proposed that the C-terminal domains in eag and erg K+ channels act as sites which drive tetramerization. From their ability to form coiled coils, these domains were referred to as tetramerizing coiled-coil (TCC) sequences. Jenke et al. also pointed out that KCNQ channels contain bipartite TCC motifs within their C-termini, exactly within the si domain, which is responsible for the subunit-specific interaction pattern. The first part of this thesis was dedicated to determine the individual role of these TCC domains on homomeric and heteromeric channel formation in order to further characterize the molecular determinants of KCNQ channel assembly. In the second part of this thesis cystein-scanning mutagenesis was employed, followed by thiol-specific modification using MTS reagents to screen more than 20 residues in the S3-S4 linker region and in the S4 transmembrane domain of the KCNQ1 channel to gain information about residue accessibility, the functional effects of thiol-modifying reagents (MTSES), and effects of crosslinking selected pairs of Cys residues by Cd+ ions, which could be used for testing model predictions based upon known Kv channel structures from the literature. According to homology modelling based on the Kv1.2 structure it was attempted to determine the proximity of individual residues from different transmembrane segments using the metal bridge approach (crosslinking by Cd+ ions). This led us to derive structural constraints for interactions between the S4 voltage sensor and adjacent transmembrane segments of KCNQ1. Similar studies have previously been performed on the Shaker K+ channel, which has served as a paradigm for structure-function research of voltage-gated K+ channels for a long time, but little is known for KCNQ channels concerning their similarity to published K+ channel structures.
Molecular mechanism of intracellular signal transduction by the angiotensin-converting enzyme
(2007)
The angiotensin converting enzyme (ACE) is an important component of the renin-angiotensin system (RAS) and is crucially involved in the homeostasis of fluid and electrolyte balance and thus in the regulation of blood pressure. The zinc metallopeptidase is involved in the generation of angiotensin II, a potent vasoconstrictor and in the degradation of bradykinin, a potent vasodilator. It is worth noting that ACE more readily hydrolyzes bradykinin than it does angiotensin I thus culminating in the net physiological effect of the production of a vasoconstrictor and the decrease in the availability of a vasodilator. ACE inhibitors have become one of the most successful therapeutic approaches as a first line of therapy in hypertension, and are also widely used in treating heart failure, myocardial infarction, stroke, coronary artery disease and impaired left ventricular function. However, one unexpected clinically relevant finding related to ACE inhibitors is their ability to delay the onset of type II diabetes that was revealed by various large clinical trials. However, the mechanisms underlying these beneficial effects of ACE inhibitor therapy are currently unclear and cannot be explained by the prevention of angiotensin II formation or the attenuated degradation of bradykinin. Thus the potential beneficial effects attributed to ACE inhibitors may occur independent of reductions in blood pressure paving way for new and/or unknown mechanism. Our group has recently redefined ACE as a signal transduction molecule which upon binding to ACE inhibitor turns on a signalling cascade leading to phosphorylation of Ser1270 by CK2, activation of JNK and changes in gene expression in endothelial cells. However the mechanism by which ACE inhibitor initiates the signalling cascade was not clear. It was hypothesized that ACE, which is anchored to the membrane with a single transmembrane domain should dimerize prior to initiating further downstream signalling events in endothelial cells. Therefore, we sought to explore whether or not ACE forms dimers in endothelial cells and whether ACE dimerization is essential for the initiation of ACE signalling in endothelial cells. Using native gel electrophoresis, we found that ACE forms dimers in endothelial cells and that there is an increase in the dimer formation upon treatment of endothelial cells with ACE inhibitors. ACE homodimerization was also demonstrated using the split-ubiquitin system and chemical cross-linking experiments. ACE dimers are also formed in endothelial cells overexpressing the non-phosphorylatable ACE, wherein ACE signalling was abolished indicating that dimerization process is not influenced by the phosphorylation of the serine residue residing in the cytoplasmic tail. Monosaccharides like glucose, galactose and mannitol did not have any influence on ACE-inhibitor induced dimerization. Making use of different monoclonal antibodies directed to the epitopes of N-domain which harbours carbohydrate recognizing domain, also did not affect dimerization. However, inactivation of the C-domain active site by introducing mutation of the key histidine residues in HEMGH consensus sequences, which complexes the zinc ions, abolished enzyme dimerization both in the basal state and in response to ramiprilat. Mutation of the C-domain also resulted in the loss of ACE inhibitor-induced ACE signalling, that is we failed to observe ramiprilat-induced increase in the phosphorylation of the Ser1270 and the subsequent JNK activation. ACE-inhibitor induced dimerization precedes the phosphorylation of Ser1270 and activation of JNK. Thus the ACE-inhibitor induced dimerization via the C-domain of ACE represents the initial step in the ACE signalling pathway which involves the activation of JNK/c-Jun pathway and leading to the changes in the gene expression in endothelial cells. Our group previously identified ACE itself as well as cyclooxygenase-2 (COX-2) as two “ACE signalling-regulated” genes. To screen for additional genes regulated in a similar manner we used DNA microarray technology, to assess ramiprilat-induced changes in the endothelial cell gene expression. 21 genes were identified to be differentially regulated of which, 7 were upregulated and 14 were downregulated by ramiprilat. However, when screened at the protein level, we found no significant differences between the untreated control cells and those treated with ramiprilat. As several other cells and tissues possess a fully functional RAS we screened plasma samples from healthy volunteers as well as from patients with coronary artery disease for the proteins identified in the microarray. We observed that the cellular retinal binding protein-1 (CRBP-1) was detectable at low levels in plasma from patients and that ramipril markedly increased serum levels of this protein. Endothelial cells overexpressing CRBP-1 demonstrated increased RXRE and PPRE activity when stimulated with 9-cis retinoic acid and rosiglitazone respectively suggesting that CRBP-1 might affect gene expression via heterodimerization of PPAR elements with RXR elements by virtue of its function as a transport protein of retinoic acid. Studies aimed at determining the consequences of elevated CRBP-1 expression on endothelial cell homeostasis are ongoing. Although the RAS has been described in many other tissues apart from endothelial cells, ACE signalling has not yet been addressed in tissues such as monocytes/macrophages, which have an increased ACE expression in an atherosclerotic setting. We observed that upon stimulation of cultured ACE expressing monocytes with ramiprilat, JNK is activated suggesting the occurrence of ACE signalling in human monocytes. It is worth noting that ACE inhibitors delay the onset of type II diabetes in spite of moderate decrease in blood pressure. To further elucidate the mechanism underlying this effect, we found that ACE inhibitors increase the PPARgamma levels in the nuclear extracts of ACE expressing monocytes which were also reproduced in human endothelial cells overexpressing human somatic ACE. However, ramiprilat did not have any direct effect on the activity of a luciferase-coupled promoter containing several copies of the PPRE in human endothelial cells. These results contrasted with the actions of the PPARgamma agonist suggesting that ramiprilat enhances PPARgamma levels through an indirect mechanism. We next hypothesized that ramiprilat might increase the levels of 15-deoxy-D12,14-prostaglandin J2 (15dPGJ2) which is a natural ligand for PPARgamma via COX enzymes in monocytes. We observed that ramiprilat was able to decrease the diminution of COX-2 levels upto 48 hours of treatment but the levels of 15dPGJ2 were too low to be detected by ELISA. However ramiprilat enhanced the plasma levels of adiponectin, a downstream target of PPARgamma, which is a anti-atherogenic and anti-inflammatory adipokine, in patients with coronary artery disease. Though adiponectin is a PPARgamma-regulated gene, the observed increase in adiponectin might be attributed to the increase in RXR rather than via PPARgamma. Taken together, the results of this investigation have revealed that ACE inhibitors initiate ACE signalling by eliciting the dimerization of the enzyme, more specifically via its C-domain active centers. The ACE signalling cascade when activated leads to the enhanced expression of ACE, COX-2 and CRBP-1 which in turn favours the heterodimerization of PPARgamma with RXR and thus results in the increased expression of “PPARgamma regulated” genes such as adiponectin. The latter results provide a molecular basis for the observation that ACE inhibitors can delay the onset of type 2 diabetes in as much as it was possible to link ramipril with CRBP-1, RXR activity and the expression of adiponectin, an adipokine associated with improved insulin sensitivity. Further work is however required to elucidate the consequences of ACE inhibitors in monocytes and adipocytes as well as in intact animals.
In this thesis we investigate the role played by gauge fields in providing new observable signatures that can attest to the presence of color superconductivity in neutron stars. We show that thermal gluon fluctuations in color-flavor locked superconductors can substantially increase their critical temperature and also change the order of the transition, which becomes a strong first-order phase transition. Moreover, we explore the effects of strong magnetic fields on the properties of color-flavor locked superconducting matter. We find that both the energy gaps as well as the magnetization are oscillating functions of the magnetic field. Also, it is shown that the magnetization can be so strong that homogeneous quark matter becomes metastable for a range of parameters. This points towards the existence of magnetic domains or other types of magnetic inhomogeneities in the hypothesized quark cores of magnetars. Obviously, our results only apply if the strong magnetic fields observed on the surface of magnetars can be transmitted to their inner core. This can occur if the superconducting protons expected to exist in the outer core form a type-I I superconductor. However, it has been argued that the observed long periodic oscillations in isolated pulsars can only be explained if the outer core is a type-I superconductor rather than type-I I. We show that this is not the only solution for the precession puzzle by demonstrating that the long-term variation in the spin of PSR 1828-11 can be explained in terms of Tkachenko oscillations within superfluid shells.
This thesis is concerned with various aspects of estimating trend output and growth and discusses and evaluates methods to prepare medium-term GDP growth projections. Furthermore, econometric techniques suited for cross-correlated macroeconomic panel data with a focus on factor models are applied for unit root and cointegration testing as well as panel error correction estimation. Applications involve the identification of growth determinants as well as the modelling of aggregate labor supply in a multi-country framework. The first chapter evaluates a very popular method for potential output estimation and medium-term forecasting---the production function approach---in terms of predictive performance. For this purpose, a particular forecast evaluation framework is developed and an evaluation of the predictions of GDP growth for the three to five years ahead for each individual G7 country is carried out. In chapter two, a new approach for estimating trend growth of advanced economies is proposed. The suggestion combines econometric methods that have been used to test and estimate the implications of the extended Solow growth model in a cross sectional time series setting with an application of multivariate time series filter techniques. The last chapter discusses several panel unit root tests designed to accommodate cross-sectional dependence. These methods are then applied to an OECD country sample of the aggregate labor supply measure "hours worked".
The formation and maintenance of a defined three-dimensional structure is a prerequisite for most proteins in order to fulfill their function in the native context. However, there are proteins, which are intrinsically unstructured and thus natively unfolded. In addition, the misfolding and aggregation of many proteins can lead to severe diseases. The investigation of non-native states of proteins significantly contributes to the understanding of protein folding and misfolding. Nuclear magnetic resonance (NMR) spectroscopy is the only known technique that can provide information on structure and dynamics of non-native states of proteins at atomic resolution. Unfolded and non-native states of proteins have to be treated as ensembles of rapidly interconverting conformers and their observed properties are ensemble and time averaged. In this thesis, hen egg white lysozyme (HEWL) and mutants thereof have been investigated by NMR spectroscopy. The reduction of its four disulfide bridges and the successive methylation of the cysteine residues renders HEWL permanently non-native (‘HEWL-SMe’). Alternatively, the exchange of the eight cysteines for alanines results in very similar states (‘all-Ala-HEWL’). Under these conditions, HEWL-SMe and all-Ala-HEWL do not resemble random coil conformations, but exhibit residual secondary and tertiary structure. The presence of hydrophobic clusters and long-range interactions around the proteins six tryptophan residues and the modulation of these properties by single-point mutants has been observed. For the NMR spectroscopic investigation, HEWL has been isotopically labelled in E. coli by expression into inclusion bodies. After purification, the 1HN, 15NH, 13Calpha, 13Cbeta, 13C’, 1Halpha and 1Hbeta resonances of HEWL-SMe and all-Ala-HEWL have been assigned almost completely using three-dimensional NMR experiments. The analysis of secondary chemical shifts revealed regions in the proteins sequence — particularly around the six tryptophan residues—with significantly populated alpha-helix like conformations. In order to further elucidate the influence of the tryptophan side chains, a set of two new pulse sequences has been developed that allowed for the successful assignment of the 13Cg, 15Ne and 1HNe resonances in these side chains. This knowledge was eventually exploited in the interpretation of two-dimensional 15N-1H photo-CIDNP spectra, which revealed a differential solvent accessibility of the tryptophan residues in all-Ala-HEWL but not in the single point mutant W62G-all-Ala-HEWL. In addition, heteronuclear R2 relaxation rates have been determined for the indole 15Ne nuclei of all-Ala-HEWL and W62G. While in the wild-type like all-Ala-HEWL, the rates are different among the six tryptophan residues, in W62G they are more uniform. Together with relaxation data from the amide backbone, these results indicate the significant destabilization of the hydrophobic clusters in the absence of W62. In contrast, in the W108G mutant the profile of the R2 relaxation rates was not found to be significantly altered. No evidence was found by R1rho relaxation rates and relaxation dispersion measurements for conformational exchange on slower (micro- to millisecond) timescales. Residual dipolar couplings have been determined for non-native HEWL in order to retrieve structural information of these states. The differences of the W62G and the wild-type like non-native HEWL is also picked up in NH-RDCs of these proteins aligned in polyacrylamide gels. Significant positive RDCs are observed in the regions of the hydrophobic clusters in all-Ala-HEWL, but to a much lesser degree in W62G. So far, all attempts to simulate RDCs from generated non-native ensembles failed even when including long-range contacts or specific phi/psi backbone angle propensities. However, the measured RDCs can be used to cross-validate structural ensembles of non-native HEWL generated by molecular dynamics simulations that are based on restraints from the other experimental data, such as the differential solvent accessibilities from the photo-CIDNP experiments and the data on the hydrophobic clustering gained from the combined mutational and relaxation studies. Finally, non-native HEWL has been investigated for the first time using two-dimensional NMR in organic solvents, which are able to induce secondary structures and ultimately lead to amyloid formation. Under these conditions severe line broadening was observed, which was attributed to exchange between different — mostly a-helical— conformations. In summary, in this thesis methods have been developed, optimized and successfully applied for the structural and dynamical characterization of non-native states of proteins and the effect of single-point mutants on the properties of such ensembles has been investigated. Data has been gained that can considerably contribute to the further elucidation of the nature of non-native states of HEWL by molecular dynamics simulations.
Leukemia inhibitory factor enhances neurogenin's pro-neural effect during mouse cortical development
(2007)
Die Entwicklung von unterschiedlichen Zelltypen waehrend der embryonalen ZNS-Entwicklung ist abhaengig von zellintrinsischen und positionsabhaengigen, aeusseren Einfluessen. Dabei bilden sich die verschiedenen Zellen in nacheinander ablaufenden bzw. sich teilweise ueberlappenden Zeitraeumen. Zuerst entstehen Radiaglia und Neuronen, nachfolgend Astrozyten und zuletzt Oligodendrozyten. Werden neurale Stammzellen/Vorlaeuferzellen (NPCs – neural precursor cells) zu unterschiedlichen Zeitpunkten entnommen und ohne den Einfluss von Wachstumsfaktoren kultiviert, so entwickeln sich diese Zellarten in der gleichen Reihenfolge. Die Neurogenese, die bei Mausembryos am Tag E11-12, nach dem Etablieren der Radialglia, beginnt, findet an E14 ihren Hoehepunkt. Zu diesem Zeitpunt werden die Gene Neurogenin1 (Ngn1) und Ngn2 in den neuralen Vorlaeuferzellen der Ventrikularzone des dorsalen Cortexes in hohem Masse exprimiert. Wie aus Untersuchungen von unserm Labor gezeigt wurde, beguenstigt es die Entstehung von Neuronen und blockiert gleichzeitig Pro-Astrozyten-Einfluesse. Zum einen inhibiert Ngn den JAK/STAT Signalweg, dessen Aktivierung fuer die Gliogenese noetig ist, indem es die Phosphoylierung von STAT1/3 auf bisher noch unbekannte Weise blockiert. Ausserdem bindet der Transkriptions-Coaktivator cAMP-response element binding protein (CBP), welches auch von den STATs fuer die Transkription benoetigt wird, bevorzugt an Ngn sobald dieses von den Vorlaeuferzellen exprimiert wird. Mit dem Tag E16 nimmt die Neurogenese in vivo wieder stark ab und es setzt die Gliogenese ein, bei der zunaechst ueberwiegend Astrozyten gebildet werden. Faktoren wie leukemia inhibitory factor (LIF) sowie ciliary neurotrophic factor (CNTF) beguenstigen dabei die Astrozytogenese indem sie den JAK/STAT Signalweg aktivieren. Die Bindung von LIF/CNTF fuehrt zur Phosphorylierung von STAT-Transkriptionsfaktoren, die ihrerseits dann an den CBP/p300 Komplex binden und schliesslich die Expression von Astrozyten-spezifischen Genen aktivieren. Die STAT-Faktoren koennen aber erst nach Abfall des Ngn-Spiegels an den Transkriptions-Coaktivator binden, da sich die Bindungsstellen dieser beiden ueberlappen. Um die Hypothese zu ueberpruefen, dass LIF auch die Neurogenese, oder spezifischer, die Wirkung von Ngn positiv beeinflusst, wurden cortikale NPCs von murinen Embryos entnommen und der Wirkung von LIF via Luciferase Assay untersucht. Dabei wurden die Vorlaeuferzellen mit Ngn und einem Reporter transfiziert, welcher den NeuroD-Promoter beinhaltete. NeuroD-Expression findet in der Regel gegen Mitte/Ende der Neurogenese statt und ist wichtig fuer die Reifung von Neuronen. Der Promoter von NeuroD beinhaltet ein E-box Element, an welches Ngn bindet und die Transkription einleitet. Wie unsere ersten Versuche zeigten, verstaerkt LIF die Transkriptionsaktivitaet von Ngn und somit die Transkription von NeuroD. Wenn aber im selben Versuch ein NeuroD-Reporter transfiziert wurde, dessen E-box mutiert war, wurde keine Transkriptionsaktivitaet gemessen, was wiederum bestaetigte, dass der pro-neurale LIF-Effekt ueber Ngn lief und E-box-Bindung noetig war. Um den Einfluss des pro-neuralen Effekts von LIF auf Proteinebene zu testen, wurden NPCs mit Ngn-Adenovirus infiziert und mit LIF stimuliert. Dabei wurden die Zellen auf die Expression von Neuron-spezifischem class III β-tubulin (TuJ1) untersucht. Die Ergebnisse zeigten, dass LIF bei Zellen, die Ngn exprimierten, die Rate der Neuronen von etwa 5% auf etwa 50% anstiegen liess, waehrend LIF bezueglich der Gliogenese (gezeigt durch die Expression von GFAP) in Ngn-exprimierenden Vorlaeuferzellen kaum Wirkung zeigte. Als naechstes sollte untersucht werden ueber welchen Signalweg LIF Ngn aktivierte. LIF bindet zunaechst an LIF receptor β (LIFRβ), der dann an glycoprotein 130 (gp130) bindet. Diese Bindung fuehrt dann zur Aktivierung mehrerer Signalkaskaden: dem JAK/STAT, dem MAPK, dem Akt/PI3K und dem PLCγ/PKC Signalweg. Da der JAK/STAT Signalweg fuer die Gliogenese wichtig ist, lag unser Fokus auf den anderen Signalwegen. Deren Aktivierung wurde dann mit spezifischen Inhibitoren blockiert und, wie auch in den Vorversuchen, die Wirkung von LIF auf Transkriptionsebene (NeuroD) in neuralen Vorlaeuferzellen bestimmt. Dabei zeigte sich, dass die Blockierung des PLCγ/PKC Signalweges die NeuroD-Promoteraktivitaet am starksten inhibierte, waehrend auch LIF´s pro-neurale Wirkung verloren ging. Dementsprechend zeigte die Western Blot Analyse, dass die Expression von class III β-tubulin (TuJ1) durch die Anwendung der PKC Inhibitoren am staerksten inhibiert wurde, wobei auch hier die Stimulation durch LIF keine erhoehte Neurogenese mit sich zog. In weiteren Versuchen konnten wir dann mit Hilfe von Immunoprezipitation demonstrieren, dass LIF die Bindung von Ngn an CBP verstaerkte (eine Bindung, welche durch PKC Inhibitoren aufgehoben wurde), was wiederum zu einer erhoehten Bindung dieses Transkriptionskomplexes an den NeuroD Promoter fuehrte, wie unsere Chromatin Immunoprezipitation (ChIP) Daten beweisen. Dies wiederum laesst darauf schliessen, dass womoeglich diese erhoehte Ngn-CBP/NeuroD-Promoter Bindung der Grund fuer die erhoehte NeuroD-Transkriptionsaktivitaet ist daher auch fuer die erhoehte neuronale Differenzierung. Interessanterweise konnten wir auch zeigen, dass Brahma-related gene 1 (Brg1), eine katalytische Untereinheit des SWI/SWF Komplexes, an den Ngn/CBP cotranscriptionalen Komplex bindet und dass diese Bindung durch LIF-Stimulation verstaerkt wurde. Dies suggeriert wiederum, dass auch Brg1 eine wichtige Rolle waehrend der murinen, cortikalen Neurogenese spielt. Dennoch, in folgenden Experimenten verblieb der Fokus auf Ngn und CBP. Um unsere Hypothese zu bestaetigen, dass PKCδ ein moeglicher Mediator des LIF-Effekts sein koennte, zeigten wir zunaechst, dass die PKCδ-Expression in cortikalen NPCs waehrend der Neurogenese erhoeht ist. Desweiteren demonstrierten wir, dass die Inhibition von PKCδ einen aehnliche Wirkung zeigte wie die Inhibition von PKC mit einem generellen PKC Inhibitor: weder war nach PKCδ-Inhibition eine LIF-induzierte NeuroD-Transkription erzielbar, noch wurde nach LIF-Stimulation der pro-neurale Marker class III β-tubulin/TuJ1 in Ngn1-infizierten NPCs exprimiert. Um aber mehr spezifisch die PKC- und PKCδ-Aktivitaet/Expression zu blockieren transfizierten wir NPCs mit PLCγ oder PKCδ siRNA. Unsere Daten zeigten hierbei, dass siRNA-transfizierte Zellen kein class III β-tubulin mehr aufweisen, was darauf hindeuted, dass PKCδ der potentielle Mediator des pro-neuralen LIF-Effekts ist. Durch unsere in vivo Daten demonstrierten wir schliesslich, dass LIF auch hierbei fuer die Neurogenese von Bedeutung ist. Verglichen wurden die Cortices von E13 LIF Het (heterozygote) und KO (knock out) Maeusen mit denen von WT (wild type) Maeusen. Durch Immunohistologie von Hirnschnitten konnten dabei keine groesseren Unterschiede bezueglich der Expression neuraler Marker beobachtet werden, waehrend aber mit Hilfe der Western Blot Analyse, eine quantitativere Methode, gezeigt wurde, dass LIF Het und KO Maeuse weniger pro-neurale Marker im Cortex exprimieren wie WT Mause. Um auch zu beweisen, dass dies auf eine verringerte Transkription von NeuroD zurueckzufuehren ist, demonstrierten wir mit Hilfe des ChIP Assay, dass LIF Het und KO Maeuse weniger Ngn1-CBP Bindung an den NeuroD-Promoter aufweisen wie WT Maeuse. Diese Experimente veranschaulichen einen eleganten Regulationsmechanismus, durch welchen ein einzelner, extrazellulaerer Faktor die unterschiedliche Differenzierung einer Zelle verstaerkt, abhaengig von der Anwesenheit oder Abwesenheit eines einzelnenn intrazellulaeren Faktors. Auch koennen durch die erlangten Resultate Strategien entworfen werden, durch die in Zukunft die Produktion bestimmter Neurone zur Heilung von verschiedenen, neurodegenerativen Krankheiten erhoeht wird.
The retinoic acid related orphan receptor alpha (RORalpha) regulates the expression of various target genes by binding to specific response elements in their promoter region. RORalpha is an interesting pharmaceutical target since it positively affects several pathophysiological processes of clinical relevance. RORalpha enhances the expression of Apo-AI protein, the major constituent of HDL, which is responsible for the cholesterol transportation. RORalpha notably contributes to the bone mineralization and generation of the extracellular bone matrix, demonstrating its involvement in osteoporosis, and by up-regulating the gene for IKBalpha, RORalpha has anti-inflammatory effects. Moreover, RORalpha is necessary for cerebellar development and the maintenance of the mammalian day-night periodicity governed by the core-clock within the suprachiasmatic nuclei. RORalpha receptors have been reported to bind cholesterol, melatonin, or to function ligand-independent. By monomeric binding to the recognition motif AGGTCA preceded by an A/T-rich sequence (ROR response element, RORE), RORalpha constitutively activates gene transcription. However, RORalpha activity is passively suppressed by its opponents RevErbalpha and RevErbbeta, which both bind to the same target sequence. ...
Many environmental chemicals are suspected of disturbing the human and animal endocrine system. These so-called endocrine disruptors can operate in many ways. The interaction of endocrine disruptive effects that eventually endanger human health is still unclear. However, one of the basic mecha-nisms of endocrine disruption is the inhibition of key enzymes in the hormone metabolism. In this study, we focused on the inhibitory potency of suspected endocrine disrupting compounds on aromatase (P450arom) and 5alpha-reductase (5alpha-Re) activities in human tissue and human cancer cells. Both enzymes are essential for the human sex steroid hormone metabolism. We were able to demonstrate that the organotin compounds tributyltin (TBT) and triphenyltin (TPT) are potent unspecific inhibitors of P450arom and 5alpha-Re activity. Prochloraz and fenarimol inhibited P450arom activity at low concentrations (IC50<2 µM), while 5alpha-Re activity was only impaired at higher concentrations (IC50>10 µM). While the human tissue assay proved to be more practical and sensitive as a screening tool for putative endocrine disruptors, the cell assay reflected partly the situation in vivo. In another experimental series, we investigated the inhibitory effect of TPT on P450arom, 5alpha-Re, 3beta-HSD type 2, 17beta-HSD type 1 and type 3 alone and in combination with the strong antioxidant dithioerythrithol (DTE). TPT inhibited unspecifically all enzymes that were tested. The experiments also showed that DTE is able to compensate the adverse effects of TPT, and that the effectiveness of the compensatory activity of DTE differs among the enzymes investigated. The suppressed 5alpha-Re activity could not be reactivated with DTE. Conceivably, cysteine residues that are responsible for the tertiary and quarternary structure of the enzyme are critical targets for TPT. A human sampling study was undertaken with the COMPRENDO partner in Gdansk. 60 Polish and 15 German blood samples were investigated for chemical residues and sex hormone concentrations. In addition, 15 placenta samples from Poland and Germany, respectively, were tested for chemical residues, P450arom activities and CYP19 mRNA contents. The chemical analysis was performed by the COMPRENDO partners in Milan (p,p´DDE), Orleans (TBT and TPT) and Ioannina (diuron, fenarimol, linuron und vinclozolin). The results showed that individual sex hormone concentrations in blood were not correlated with chemical body burden. The detected differences in sex hormone concentrations, specific aromatase activity and relative CYP19 mRNA content of Polish and German donors were presumably the result of other factors than the ones determined in this study. Another task of the EU-project was the investigation of the effects of chemical exposure of the aquatic model organisms Pimephales promelas, Rutilus rutilus and Xenopus laevis. We investigated the specific P450arom and 5alpha-Re activities in brain and gonads of the animals. During the qualitative investigation of the androgen metabolism in Xenopus laevis brain, 5alpha-reductase activity was discovered for the first time. In contrast to the inhibitory potency of TPT discovered in our enzyme assays, TPT exposure of aquatic model organisms had no observed effect on enzyme activity in the organs investigated, except for P450arom activities in female gonads of Pimephales promelas at 320 ng TPT/L. In this group, mean P450arom activities were elevated, possibly as a result of an overshooting upregulation due to the inhibition of P450arom by TPT. The exposure of Rutilus rutilus and Xenopus laevis to the effector substances methyltestosterone and letrozole resulted in slightly different mean enzyme activities compared to the control group. In conclusion, many of the tested pesticides are able to inhibit P450arom and 5alpha-Re, and thus might be of clinical relevance. However, results are not always coherent, and possible risks for human and wildlife health are therefore difficult to predict. Risk assessment will require large studies with an additional number of short and long term in vitro and in vivo assays. Any extrapolation to humans should be very meticulously performed.
The following thesis is concerned with the elucidation of structural changes of RNA molecules during the time course of dynamic processes that are commonly denoted as folding reactions. In contrast to the field of protein folding, the concept of RNA folding comprises not only folding reactions itself but also refolding- or conformational switching- and assembly processes (see chapter III). The method in this thesis to monitor these diverse processes is high resolution liquid-state NMR spectroscopy. To understand the reactions is of considerable interest, because most biological active RNA molecules function by changing their conformation. This can be either an intrinsic property of their respective sequence or may happen in response to a cellular signal such as small molecular ligand binding (like in the aptamer and riboswitch case), protein or metal binding. The first part of the thesis (chapters II & III) provides a general overview over the field of RNA structure and RNA folding. The two chapters aim at introducing the reader into the current status of research in the field. Chapters II is structured such that primary structure is first described then secondary and tertiary structure elements of RNA structure. A special emphasis is given to bistable RNA systems that are functionally important and represent models to understand fundamental questions of RNA conformational switching. RNA folding in vitro as well as in vivo situations is discussed in Chapter III. The following chapters IV and V also belong to the introduction part and review critically the NMR methods that were used to understand the nature and the dynamics of the conformational/structural transitions in RNA. A general overview of NMR methods quantifying dynamics of biomolecules is provided in chapter IV. A detailed discussion of solvent exchange rates and time-resolved NMR, as the two major techniques used, follows. In the final chapter V of the first part the NMR parameters used in structure calculation and structure calculation itself are conferred. The second part of the thesis, which is the cumulative part, encompasses the conducted original work. Chapter VI reviews the general NMR techniques applied and explains their applicability in the field of RNA structural and biochemical studies in several model cases. Chapter VII describes the achievement of a complete resonance assignment of an RNA model molecule (14mer cUUCGg tetral-loop RNA) and introduces a new technique to assign quaternary carbon resonances of the nucleobases. Furthermore, it reports on a conformational analysis of the sugar backbone in this RNA hairpin molecule in conjunction with a parameterization of 1J scalar couplings. Achievements: • Establishment of two new NMR pulse-sequences facilitating the assignment of quaternary carbons in RNA nucleobases • First complete (99.5%) NMR resonance assignment of an RNA molecule (14mer) including 1H, 13C, 15N, 31P resonances • Description of RNA backbone conformation by a complete set of NMR parameters • Description of the backbone conformational dependence in RNA of new NMR parameters (1J scalar couplings) Chapters VII & VIII summarize the real-NMR studies that were conducted to elucidate the conformational switching events of several RNA systems. Chapter VIII gives an overview on the experiments that were accomplished on three different bistable RNAs. These molecules where chosen to be good model systems for RNA refolding reactions and so consequently served as reporters of conformational switching events of RNA secondary structure elements. Achievements: • First kinetic studies of RNA refolding reactions with atomic resolution by NMR • Application of [new] RT-NMR techniques either regarding the photolytic initiation of the reaction or regarding the readout of the reaction • Discovery of different RNA refolding mechanisms for different RNA molecules Deciphering of a general rule for RNA refolding methodology to conformational switching processes of RNA tertiary structure elements. The models for these processes were a) the guanine-dependent riboswitch RNA and b) the minimal hammerhead ribozyme. Achievements: • NMR spectroscopic assignment of imino-resonances of the hypoxanthine bound guanine-dependent riboswitch RNA • Application of RT-NMR techniques to monitor the ligand induced conformational switch of the aptamer domain of the guanine-dependent riboswitch RNA at atomic resolution • Translation of kinetic information into structural information • Deciphering a folding mechanism for the guanine riboswitch aptamer domain • Application of RT-NMR techniques to monitor the reaction of the catalytically active mHHR RNA at atomic resolution In the appendices the new NMR pulse-sequences and the experimental parameters are described, which are not explicitly treated in the respective manuscripts.
All living organisms exhibit daily fluctuations in biochemical, physiological and behavioural parameters driven by endogenous oscillators, residing in the organism itself. In mammals, the core circadian oscillator is located in the paired suprachiasmatic nuclei (SCN) of the hypothalamus. Circadian rhythm generation in the SCN depends upon the expression of clock genes interacting in positive and negative transcriptional/translational feedback loops. The SCN governs the timing of peripheral circadian oscillators by neuronal pathways and by neuroendocrine mechanisms. An important neuroendocrine hand of the core circadian oscillator is melatonin, which is produced in and secreted from the pineal gland night by night. The adenohypophysis represents a peripheral circadian oscillator and the secretion of one of its hormones, prolactin, is known to be regulated by melatonin. The aim of the present study was to analyze a putative influence of melatonin on the activity state and diurnal variations of identified cell types in the hypophysis. Particular attention was paid to lactotroph, gonadotroph and pars intermedia cells. Experiments were performed with young male mice of different strains: melatonin-proficient C3H, melatonin-deficient C57BL, melatonin-proficient C3H with targeted deletions of the Mel1a receptor (MelaaBB), Mel1b receptor (MelAAbb) or both receptors (Melaabb). Cells producing prolactin (PRL), follicle stimulating hormone (FSH) were immunocytochemically identified and the presence of phosphorylated CREB protein (pCREB) and clock gene protein PER1 was demonstrated by double immunolabeling at different time points during the light/dark cycle in melatonin deficient, melatonin proficient and melatonin receptor knockout mice. Melatonin influence on Prl mRNA levels was investigated by means of in situ hybridization. At night the percentage of lactotroph cells showing a positive nuclear pCREB- and PER1-immunoreaction is significantly smaller in C57BL than in C3H mice. In both mouse strains, the percentage of pCREB –immunoreactive cells is minimal in the early morning and gradually increases to reach a maximum in the late night. PER1 levels show a parallel temporal variation in C3H, but in C57BL, they are drastically reduced in the early afternoon. The percentage of FSH-immunoreactive cells showing pCREB immunoreaction was significantly lower in the melatonin-deficient C57Bl mice than in the melatonin-proficient C3H mice during the second part of the day and during the night. In each strain, the percentage of FSH-immunoreactive cells was lowest at the early morning and gradually increases until the maximum at late night. In wild type (MelAABB) and MelAAbb mice the percentage of lactotroph cells with nuclear pCREB immunoreactions varied significantly over 24 h period, whereas in MelaaBB and Melaabb mice no significant differences were found between the five time points analyzed. The number of Prl mRNA expressing cells was significantly higher in MelaaBB and MelAAbb than in their wild type (MelAABB) littermates. pCREB levels in the pars intermedia did not show rhythmic variation in wild type or Melaabb animals, but wild type mice had higher pCREB levels than Melaabb. The observation that, during darkness, the percentage of lactotroph cells with nuclear pCREB immunoreaction is significantly higher in C3H than in C57BL mice suggests the existence of a distinct cell population that is under the control of melatonin-dependent intrapituitary signaling. Results with melatonin receptor knockout mice indicate that Mel1a and Mel1b melatonin receptors are involved in the control of the activity state of lactotroph cells, but to a differing degree. Analysis of cells expressing Prl mRNA showed that inhibitory action on the Prl expression is mostly mediated through the Mel1a receptor. The significant difference between pCREB immunoreaction in gonadotroph cells of C3H and C57BL mice might suggest that, like lactotrophes, FSH cells represent a heterogeneous population and only a subpopulation is under control of melatonin signaling. The present study is first to show that melatonin signaling also affects pCREB levels in pars intermedia of mice.
To determine the effects of inhaled IL-10 at different doses and different time points on the pulmonary and systemic inflammatory response during endotoxemia, 48 ventilated, anaesthetized rats (mean body weight ± standard deviation, 500 ± 33g) were randomly assigned to six groups (n = 8, each). Interleukin-10 was nebulised either prior to or following the intravenous injection of LPS (5mg/kg) at two doses (5.0 mycro-g or 0.5 mycro-g) in our groups. Eight rats received the same insult with no further treatment (LPS-only group). Another eight rats served as controls without endotoxemia but with aerosolized phosphate-buffered saline, the solvent of IL-10 (Sham group). Concentrations of TNF-alpha, IL-1beta, IL-6, and IFN-gamma were analyzed in plasma and bronchoalveolar lavage fluid (BALF). In addition, the nitrite release from ex-vivo cultured alveolar macrophages was determined. As compared to the LPS-only group, the concentrations of the proinflammatory cytokines TNF-alpha, IL-1beta, IL-6, and IFN-gamma in plasma were significantly reduced in the group, which inhaled 5 mycro-g IL-10 before LPS injection (p< 0.0125). Spontaneous nitrite release from exvivo cultured alveolar macrophages was suppressed in this group (p< 0.0125). Inhalation of 0.5 mycro-g IL-10 before LPS injection and both dosages of IL-10 inhalation (5 mycro-g or 0.5 mycro-g) after LPS injection did not significantly influence either inflammatory cytokine concentrations in BALF, in plasma or the nitrite release from ex-vivo cultured alveolar macrophages. In this study, inhaled IL-10 only demonstrated anti-inflammatory effects when it was administered at 5 mycro-g prior to the induction of experimental endotoxemia. Interleukin-10 aerosol had no effect when it was given either following induction of endotoxemia or given at a lower dosage (which here was 0.5 mycro-g) either before or following injection of lipopolysaccharide.
Nicotinic acid has been used in the clinical treatment of elevated blood lipid levels for over 50 years. Although it has a beneficial effect on myocardial infarction and blood lipid profiles, its widespread use has been hampered by side effects such as skin rashes and a burning sensation on the upper body. Since elevated blood lipid levels, especially ones of VLDL and LDL cholesterol are a frequent indication and high risk factor for coronary and cardiac diseases, finding a compound with an enhanced pharmacological profile, still holding the desired effects, but without inconvenient side effects, is a very appealing aim to many pharmaceutical companies. These efforts have already produced two marketed drugs, Acipimox and Acifran, but they have not been able to overcome the restrictions already imposed on the treatment by nicotinic acid. Although proposed long before, in the year 2000 the gene for the nicotinic acid receptor in mouse PUMA-G was cloned, and in 2003 the discovery of the genes HM74 and HM74A followed, which comprise the homologous low and high affinity receptors for nicotinic acid in humans. The discovery of this G Protein-coupled receptor target allowed a more directed approach for the search of alternative compounds. This work is the first report of the heterologous overexpression of the high affinity GPCR gene HM74A in the methylotrophic yeast Pichia pastoris. The protein product, NAR1, was pharmacologically characterized, and displayed a binding affinity of 224.8 nM to its ligand nicotinic acid, showing a similar activity profile compared to those displayed in human tissue, which were determined to be 60 nM to 90 nM. Additionally, inhibitory constants (Ki) for Acifran and Acipimox were determined to be 4.5 µM and 50.5 µM, respectively. Furthermore, the total yield of NAR1 reached 42 pmol/mg membrane protein, which corresponds to 0.4 mg of receptor produced per liter yeast culture, opening up the perspective of large scale protein production to facilitate high throughput screening drug discovery efforts and structural studies. In addition, NAR1 could be solubilized in n-decyl-β-D-maltopyranoside and purified to homogeneity after immobilized metal affinity chromatography and a second affinity chromatography step on immobilized monomeric avidin, yielding a single peak on gel filtration, while the purified receptor was able to bind ligand, as shown in NMR Saturation Transfer Difference (STD) measurements. It could be shown that NAR1 is desensitized by β-arrestin 1 in vivo in confocal microscopy studies on HEK and BHK cells. This finding provides a native binding partner for the stabilization of the receptor upon solubilization and purification. Finally human β-arrestin 1 could be produced as a constitutively active variant, comprising residues 1-382 in Pichia pastoris and Escherichia coli. The purified protein was used for in vitro binding experiments and shown to be capable of interacting with NAR1. Although the interaction and formation of the complex was only possible to a limited extent, it leaves open the perspective of crystallizing NAR1 in its active conformation, bound to nicotinic acid and β-arrestin 1.
Das genetische Material der Zellen besteht aus Molekülketten der Desoxyribonukleinsäure (DNA), die ein Träger der Erbinformation ist. In normalen Körperzellen wird die Erbinformation der DNA in eine andere Molekülkette, die sogenannte Ribonukleinsäure (RNA), übersetzt. Die RNA reguliert die Bildung von neuem Protein in der Zelle. Dass die RNA nicht bloß ein „Stempel“ ist, der die Informationen der DNA weitervermittelt, darin sind sich die Experten heute einig. RNA-Moleküle können Informationen speichern, katalytische Aktivitäten entfalten, sich perfekt tarnen, und sie regulieren auch als Produkt ihre eigene Synthese. Manche Viren enthalten ebenfalls RNA (oder DNA) und können so den Produktionsapparat der Zelle täuschen. Erkenntnisse über die Wechselwirkung dieser RNA mit natürlichen und synthetischen Liganden können zur Suche nach potentiellen Wirkstoffen beitragen. Nukleinsäuren sind lineare Biopolymere von grundlegenden Untereinheiten, die Nukleotide genannt werden und aus Adenin (A), Cytosin (C), Guanin (G), Urazil (U), und Thymin (T) zusammengesetzt sind. Sie sind jedoch in der Lage sich zu falten und so eine Doppel-Helixstruktur auszubilden. Diese besteht größtenteils aus den bekannten "Watson-Crick-Basenpaaren" (G-C und A-U oder A-T), die zur Stabilität der Struktur beitragen, sowie aus den weniger stabilen G-U-Paaren. Durch die Wechselwirkung zwischen verschiedenen Sekundärstrukturelementen entstehen Tertiärstrukturelemente, deren Struktur und Dynamik oft nur schwer experimentell zu bestimmen sind. Fortschritte in der RNA-Strukturanalyse wurden durch Röntgenkristallographie und Kernresonanzspektroskopie (NMR) möglich. Durch die Röntgenkristallographie wurden viele RNA-Eigenschaften festgestellt. Allerdings besteht keine Kristallstruktur für alle mögliche Einzelnfaser-RNA-Haarnadeln, weil diese immer dazu neigen, in eine linearen doppelte Faserform zu kristallisieren, die geringe biologische Bedeutung hat. Außerdem wurde mit Hilfe der NMR-Spektroskopie das dynamische Verhalten von RNA, z.B. Entfaltungsprozesse bei ansteigender Temperatur, beobachtet. Jedoch erlauben diese experimentellen Daten oft keine direkte mikroskopische Beschreibung der molekularen Prozesse. Molekulardynamik (MD)-Simulationen von biologischen Systemen ermöglichen es hingegen, diese Prozesse in atomischem Detail zu untersuchen. Die MD-Simulation beschreibt ein molekulares System auf atomarer Ebene mit Hilfe der klassischen Mechanik. Kräfte werden von empirischen Potentialen abgeleitet. Sie liefern zeitabhängige Trajektorien, die sich aus den Newton'schen Bewegungsgleichungen ergeben. Durch verbesserte Computerleistung, bessere Kraftfelder, und neu entwickelte genauere Methoden stimmen heutzutage MD-Simulationen von RNA mit experimentellen Daten immer besser überein. In meiner Doktorarbeit wurden MD-Simulationen durchgeführt um die Dynamik, die Struktur und insbesondere die Stabilität von RNA-Hairpins theoretisch zu beschreiben, um so ein erweitertes Verständnis für die dynamischen Vorgänge zu erhalten. Auch der SFB 579 der Universität Frankfurt beschäftigt sich mit RNA-Systemen. Erforscht wird unter anderem der D-Loop des Coxsackievirus B3 (CVB3), der Virenmyocarditis verursacht. Die Interpretation dieser experimentellen Daten wird durch MD-Simulation möglich. In dieser Arbeit wurden das GROMACS Software-Paket und das AMBER Kraftfeld verwendet, um das strukturelle, dynamische und thermische Verhalten der RNA-Hairpins mit Hilfe von MD-Simulationen auf atomarer Ebene zu untersuchen. Betrachtet wurden die 14-mer RNA-Hairpins, uCACGg und cUUCGg. Die verfügbaren NMR-Strukturen zeigen, dass das uCACGg-Tetraloop auffallend ähnlich in der gesamten Geometrie und den Wasserstoffbindungen zu der experimentellen Struktur des cUUCGg-Tetraloop ist, obwohl die schließende Basenpaarsequenz der beiden Tetraloops unterschiedlich sind. Trotz beachtlicher struktureller Ähnlichkeit unterscheiden sich allerdings die uCACGg und cUUCGg Tetraloops in Funktionalität und Thermostabilität. Zunächst orientiert sich unser erstes Bemühen an der Frage nach einem guten Modell für RNA-Hairpins und Simulationsbedingungen, um die zu untersuchenden RNA-Hairpins in Wasser möglichst realitätsnah zu simulieren. Erstens werden drei Versionen des biomolekularen AMBER-Kraftfelds geprüft, indem man die 60 ns Simulationen des 14-mer uCACGg-Hairpins durchführt. Die simulierten strukturellen Eigenschaften und Atomfluktuationen zeigen hohe Ähnlichkeiten in den drei Kraftfeldern. Darüber hinaus stimmen die von MD-Simulationen berechneten Atomkernabstände mit den experimentellen NMR-Daten gut überein. Die gute Übereinstimmung zwischen den Simulationen und den strukturellen NMR Daten belegt die Fähigkeit des AMBER-Kraftfelds zur Beschreibung der strukturellen Eigenschaft von kleinen RNA-Hairpins. Anschließend werden die Einflüsse der Methoden, welche die langreichweitigen, elektrostatischen Wechselwirkungen beschreiben, auf die strukturellen Eigenschaften untersucht. Insbesondere werden die Ergebnisse der Reaktionfeld-Methode mit denen der Particle Mesh Ewald (PME)-Methode verglichen. Es zeigt sich, dass die PME-Methode die elektrostatischen Wechselwirkungen am besten beschreibt, auch wenn die Simulationen der beiden Methoden Ähnlichkeit in der Struktur-Stabilität und der Atomfluktuation bei niedriger Natriumkonzentration aufweisen. Drittens wird der Kationseffekt auf die RNA-Stabilität untersucht. Betrachtet wurden zwei unterschiedliche Kationen (ein- und zweiwertig) und verschiedene Konzentrationen. Die Simulationen weisen darauf hin, dass sich die Metallionen in der Affinität zum RNA-Hairpin unterscheiden, wenn Na+ und/oder Mg2+ als Gegenionen verwendet werden. Weiterhin wird gezeigt, dass sich die bevorzugten Positionen der Na+-Ionen in der großen Furche (major groove) des RNA-Hairpins befinden. Insbesondere die Anlagerungsort der Na+-Ionen liegen in der Nähe des schließenden Basenpaar U5-G10. Im Vergleich zu Na+-Ionen lagern sich Mg2+-Ionen sowohl an die RNA-Basen U3, A4-U11, und die Phosphat-Gruppe, als auch an das schließenden Basenpaar U5-G10 an. Bestätigt werden die Modelle und Simulationsbedingungen durch den Vergleich von Parametern, die sowohl experimentell als auch durch Simulationen ermittelt werden können. Ferner erlauben MD-Simulationen Einblick in das System, indem sie detallierte Konformations- und andere Verteilungen liefern. In der vorliegenden Arbeit wurden die Einflüsse der Loopsequenz und des schließenden Basenpaares auf die Verteilung der Konformationen, der internen Bewegungen, und auf die Thermostabilität von zwei RNA-Hairpins mit Hilfe dieser Modelle untersucht. Zunächst wurden die strukturellen Eigenschaften bei Raumtemperatur ausgewertet. Die starken strukturellen Ähnlichkeiten und die gute Übereinstimmung mit NMR-Daten bestätigen die Hypothese, dass die zwei Tetraloops zur gleichen “erweiterten” RNA-Familie gehören. Diese zwei Hairpins haben ähnliche Lösemittelzugängliche Oberflächen (solvent accessible surface), wobei deren Lösemittel zugänglichen funktionellen Gruppen unterschiedlich sind. Weiterhin weist das uCACGg-Hairpin eine stärkere Tendenz auf Wasserstoffe abzugeben als das cUUCGg-Hairpin, was in den unterschiedlichen Bindungsaffinitäten zwischen diesen Hairpins und der viralen Protease begründet liegt. Darüber hinaus wurde der Faltungs- und Entfaltungsprozess mit Hilfe der Replica-Exchange-Molekulardynamik-Simulationen untersucht. Diese Untersuchung zielt auf das bessere Verständnis der unterschiedlichen Thermostabilität der Hairpins, indem sie die möglichen Zwischenprodukte im atomaren Detail liefern. Sowohl experimentell als auch von den MD-Simulationen ergibt sich eine Differenz in den Schmelztemperaturen der beiden Hairpins von ungefähr 20 K. Allerdings sind die von MD beobachteten Schmelztemperaturen 20 % höher als die von Experiment zu ansehende Wert. Die Ergebnisse machen deutlich, dass die Schmelztemperaturdifferenz nicht auf die Unterschiede in der Sequenz, in der Struktur, oder in der Dynamik der Loops zurückführen sind, sondern auf die Unterschiede der Basenpaaren in den Stämmen. Weiterhin wird gezeigt, dass sich das uCACGg-Hairpin einerseits kooperativ entfaltet, und die Entfaltung des cCACGg-Hairpins anderseits weniger kooperativ stattfindet. Um die schnelle interne Dynamik der uCACGg- und cUUCGg-Hairpins zu untersuchen, erlauben die Simulationen von 50 ns eine akurate Beschreibung der schnellen internen Bewegung der RNA-Hairpin, obwohl der den Hairpins zugängliche Konformationsraum nicht vollständig abgedeckt wird. Die NMR-Relaxationsparameter, die mit Hilfe der MD-Simulationen zurückgerechnet wurden, bestätigen das Modell und die Simulationsbedingungen der MD-Simulationen. Im Hinblick auf die Übereinstimmung kann man den besten Ansatz zur Berechnung der NMR-Ordnungsparameter bestimmen. In dieser Arbeit wurden drei verschiedene Ansätze angewandt, nämlich das Fitting von 100 ps auf modellfreiem Ansatz nach Lipari-Szabo, equilibrium average, und das Gaussian Axial Fluctuation (GAF)-Modell. Die zwei letzteren können nur qualitativ mit den experimentellen Daten übereinstimmen. Die NMR-Ordnungsparameter können mit Hilfe des Modells von Lipari-Szabo richtig ermittelt werden, wenn sich die interne Bewegung in kleineren Zeitskalen als zur Gesamtbewegung vollzieht. Vorausetzung für die Berechnung dieses Modells ist aber, dass das Fitting der internen Korrelationsfunktionen nur auf den ersten Teil von 100 ps der Korrelationsfunktionen eingesetzt wird. Die berechneten Ordnungsparameter deuten auf ein unterschiedliches Verhalten der beiden Hairpins besonders im Loop-Bereich hin. Die konformationelle Umordnung, die beim UUCG-Loop beobachtet wurde, tritt beim CACG-Loop nicht ein. Zusammenfassend lässt sich sagen, dass es durch den Einsatz von MD Simulationen ermöglicht wird, die strukturellen und dynamischen Eigenschaften der RNA-Systeme auf atomarer Ebene zu untersuchen. Als Schlussfolgerung zeigt diese Doktorarbeit, dass sich die Studie der konformationell Dynamik der RNA-Systeme durch die Kombination aus MD-Simulation und NMR-Spektroskopie sowie der Leistungsfähigkeit der MD-Simulationen, die die interne Bewegungen deutlich beschreiben können, untersuchen lässt.
Two types of proteins transport ions across the membrane – ion channels and ion pumps. Ion pumps transport ions against their electrochemical gradient by co-transporting another ion or a substrate molecule through a concentration gradient or by coupling this process to an energy source like ATP. Those that couple ATP hydrolysis to ion transport are called ion motive ATPases and can be classified as ‘V’, ‘F’ and ‘P’ types. In this thesis, two sub-classes of P-type ATPases, PIIIA and PIB were studied. Attempts were made to over-express and crystallize the plant proton pump AHA2 (a PIIIA-ATPase). Also, the two putative copper transporting ATPases, CtrA3 (CopB-like) and CtrA2 (CopA-like) from Aquifex aeolicus (both PIB pumps) were over-expressed in E. coli and characterized. PIIIA-type pumps transport protons across the membrane and are found exclusively in plants and fungi, and probably some archaea. One of the most characterized proton pump biochemically is the A. thaliana proton pump AHA2. An 8Å projection map of this enzyme is already available (Jahn 2001). PIBATPases, also called CPX type pumps transport heavy metal ions such as Cu+, Cu2+, Zn2+, Pb2+, Cd2+, Co2+ across biological membranes and play an important role in homeostasis and biotolerance of these metals. CopA and CopB are two such proteins that transport copper across cell membrane found in many prokaryotes. CopB-like proteins are found almost exclusively in bacteria, with CPH sequence motif, while CopA-like proteins have CPC sequence motif, also found in eukaryotic copper transporters including human ATP7A and ATP7B. CopB extrudes Cu2+ across the membrane. CopA is activated by and transports Cu+ but the direction of transport is debated. Attempts were made to over-express the plant proton pump AHA2 in yeast Pichia pastoris. However, the yeast expressed only a truncated protein, which could not be used for further studies. It can be concluded that P. pastoris strain SMD1163 is not a good host for expression of AHA2. Focus was then shifted to AHA2 that has been over-expressed and purified from S. cerevisiae strain RS72. Growth and purification protocols had to be changed from published methods because of laboratory constraints and this probably had an effect on the protein produced. The protein purified from S. cerevisiae could not be crystallized reproducibly for structural studies by electron microscopy. CtrA3 was expressed in E. coli and purified using Ni2+-NTA matrix. Like CopB of A. fulgidus (Mana Capelli 2003), it was active only in the presence of Cu2+ and to some extent in Ag+. The protein was maximally active at 75°C, at pH 7 and in presence of cysteine. Lipids were essential for the activity of CtrA3. However, when the protein was purified in Cymal-6, CtrA3 could not hydrolyze ATP, even when lipids were added to the reaction mixture. For reconstitution of CtrA3 into liposomes for 2D crystallization, several lipids were tested. To screen the lipids compatible for protein incorporation, CtrA3 was dialyzed with different lipids at a high lipid-to-protein ratio of 10:1 and centrifuged by sucrose density gradient. Protein incorporated in lipids localized with liposome fraction in the gradient. Most of the CtrA3 was incorporated into DPPC with no aggregation. This lipid was used for reconstitution of CtrA3 at low LPRs, and at an LPR of 0.3-0.5, the protein formed 2D crystals. A NaCl concentration of 50mM was necessary for the formation of crystals. However, salt removal by dialysis prior to harvesting was essential for obtaining wellordered lattices of CtrA3. Addition of preservatives like trehalose and tannin or direct plunging in liquid ethane for cryo-microscopy destroyed the crystal lattice. Similar to CtrA3, the gene responsible for expression of CtrA2 was amplified from genomic DNA of A. aeolicus and expressed in E. coli and purified by Ni2+-NTA. Functional characterization of CtrA2 was done by analyzing ATP hydrolysis activity of the enzyme. Similar to CopA of A. fulgidus (Mandal 2002), CtrA2 was activated in the presence of Ag+ and to some extent, Cu+. It is possible that both the copper ATPases of A. aeolicus have different ion selectivity- CtrA3, specific for Cu2+ and CtrA2, specific for Cu+. Maximal activity of CtrA2 was also at 75°C. Cysteine was essential for activity of CtrA2, but the protein was not dependent on addition of lipids for activation. Reconstitution of CtrA2 was done similar to CtrA3 for screening of lipids for 2D crystallization. Of the lipids tested, DOPC reconstituted the protein best. However, screening at low LPRs did not yield any crystals. Even though both CtrA3 and CtrA2 are similar heavy metal transporting Ptype ATPases from the same organism and have 36% identity, they behaved completely different in their expression levels in E. coli, purification profiles, activity and reconstitution in lipids.
Purification and characterization of heterologously produced cannabinoid receptor 1 and G proteins
(2007)
G protein coupled receptors form the largest group of transmembrane proteins, which are involved in signal transduction and are targeted directly or indirectly by 40-50% of the drugs in the market. Even though a lot of biochemical and pharmacological information was acquired for these receptors in the past decades, structural information is still insufficient. G protein coupled receptors are expressed in a very minute scale in the tissues. Purification of G protein coupled receptors, in amounts needed for structural studies, from native tissue is tedious and almost impossible. To overcome this first hurdle of insufficient protein, several heterologous protein expression systems are being used. Another difficulty in structural determination of a G protein coupled receptor is that it is a membrane protein. Membrane proteins are difficult targets for structural studies. One of the possible reasons is the little hydrophilic surface area on the membrane protein, reducing the chances of crystal contact between the molecules. The present work is an attempt to investigate possible ways to overcome these problems. Aim of the project was to use G proteins to increase the hydrophilic area of the G protein coupled receptor. G protein is a physiological partner to the G protein coupled receptor which makes the complex functionally relevant. In the present work five G alpha proteins were purified to homogeneity by a two step purification using metal affinity and ion-exchange chromatography. The G alpha subunits purified were tested for their detergent susceptibility. It was found that only some G proteins were active in the presence of detergent. Observation from contemporary reports also suggest that the G alpha proteins expressed in Escherichia coli, alone may not be sufficient to bind to the G protein coupled receptors in solution. So the project was extended towards expressing a G protein coupled receptor which was reported to exist in a complex with the G proteins, in the cells. Purifying such a functional complex could be more beneficial to use for crystallization. Cannabinoid receptors were chosen for heterologous expression and purification. Production of recombinant cannabinoid receptor 2 was investigated in Pichia pastoris. The protein obtained was highly heterogenous. There were several oligomeric forms as well as degradation products in the cell membranes. Most of the protein was lost in the purification steps leading to a poor yield. Several oligomeric forms and other impurities were still present in the protein sample after purification. Alternatively, a baculovirus mediated insect cell expression system was investigated, to produce the receptors. Cannabinoid receptor 1 was investigated in insect cell expression system because of its better biochemical understanding and pharmacological importance than cannabinoid receptor 2. Cannabinoid receptor 1 was produced in two forms, a full length and a distal carboxy terminal truncated version. All the several gene constructs made could be expressed in the Spodoptera frugiperda (Sf9) insect cells. Expression levels (Bmax) for the constructs with a decahistidine tag at the amino terminus and Strep-tagII at the carboxy terminus were 40 pmol/mg and 53 pmol/mg respectively, for full length and truncated versions. These expression levels are 2 fold higher than the levels reported till now in the literature. As was quite evident from previous experiences of other research groups, purification of this receptor was a challenge. Protein purified from immobilized metal affinity chromatography (Ni-nitrilo tri acetate)(Ni-NTA) was not even 50% pure. A second purification by immobilized monomeric avidin or Streptactin agarose, making use of Biotag and StreptagII respectively, drastically reduced the protein recovery. Later on, purification of receptor was investigated on different metal chelating resins. His-Select, a Ni-NTA based matrix from Sigma, with much lesser density than Ni-NTA from Qiagen, showed a better purification profile. Purification was optimized to get 80% homogeneity but with low yield (20%). Further efforts are needed to improve the yield and purity of the receptor, to use it for crystallization. Cannabinoid receptors are known to exist in a precoupled form to G proteins in the cells. The existence of such precoupled forms of the receptor was investigated using the fluorescence techniques. Guanosine-5-triphosphate binding assay on the cell membranes, in the absence of agonists confirmed the active precoupled form of the receptor. It was found that it is possible to co-immunoprecipitate the complex. These results show that the truncated cannabinoid receptor can be produced in functional form in insect cells in much higher yields than reported. This receptor exists as a complex with G proteins even in the absence of ligands. It was also shown that the receptor/G protein complex can be coimmunoprecipitated. Further work is required to investigate the possibility of purifying this complex to use it for co-crystallization.
G-protein coupled receptors (GPCRs) comprise the largest superfamily of cell surface receptors and possess a signature motif of seven transmembrane helices. The endothelin B (ETB) receptor is a member of rhodopsin like GPCR family. It plays an important role in vasodilation and is found in the membranes of the endothelial cells enveloping blood vessels. Knowledge of the three-dimensional structure of G-protein coupled receptors in general would significantly add to our understanding of their molecular mechanisms and would be useful in the search for new specific drugs. However, three-dimensional structural analysis will require milligram quantities of pure and homogeneous protein. This dissertation is a study of the production, biochemical characterization and preliminary structural studies of the human ETB G-protein coupled receptor. The present work aimed at elucidating the structure and mechanistic details of function of the receptor by using a combination of X-ray crystallographic and NMR methods for collecting structural data. To obtain homogenous and monodisperse receptor protein preparation for structural and functional studies, we implemented the baculovirus expression system for the production of ETB receptor for the present work. The two step affinity purification ensured capture of full-length receptor. Silver stained SDS-PAGE of the purified receptor-ligand complex indicated greater than 90% protein purity. Based on previous reports, we used the high affinity ligand (endothelin -1) binding to the receptor for co-crystallization of receptor-ligand complex by locking the receptor in the activated conformation. As a prerequisite for 3D crystallization trials, the stability of the detergent solubilized receptor-ligand complex was assessed with respect to pH, temperature and time. Receptor-ligand complex did not show any degradation and aggregation over 6 days at 4°C and 18°C. Interestingly, change of pH suggested that receptor-ligand complex is unstable at lower pH due to possible charge induced conformational changes. In our work, we introduced the idea of using fluorophore labeled ligand for simple visual recognition of the receptor-ligand complex during purification and crystallization. On the other hand, we alternatively used biotinylated endothelin-1 to produce an adequate amount of ligand bound receptor complex, thus ensuring homogeneity of the purified complex for use in structural studies. Thus far, preliminary crystals have been obtained for both the unlabelled ET-1 and fluorophore labeled ET-1 complexed with ETB receptor. Moreover, we performed the systematic investigation of the protein/peptide binding partner for the receptor-ligand complex with the chief aims of stabilizing structure and increasing the possibilities of 3D-crystal contacts. Thus subsequent to formation of receptor-ligand complex, the additional in vitro formation of a ternary arrestin-receptor-ligand complex was also attempted for use in structural studies. We successfully demonstrated that arrestin mutant (R169E) forms a tight complex with ETB receptor regardless of its phosphorylation state. A second approach to get insight into the ETB receptor ligand binding site relied on the use of spin isotope labeled ET-1 ligand peptide by employing solid state MAS NMR method. Preliminary data provided compelling evidence that the C-terminal region of the peptide is immobilized in an ordered environment and presumably bound to the receptor. This indicates that the approach is feasible, although there are difficulties in sample preparation for further spectral measurements and data collection which are currently being discussed in ongoing investigations. At this point of our research work, we initiated a collaborative effort to obtain high yields of pure, active receptor without post translational modifications, from an E. coli cell lysate based in vitro expression system. We successfully optimized the production of homogenous and monodisperse endothelin B receptor in mg amounts. Thus this could potentially provide an alternative source of high quality receptor production in large quantities for immediate crystallization trials. Thus we hope that the results from these investigations can be applied in a more general sense to the production and crystallization of other G protein-coupled receptors.