Novel Insights into PKG Activation and cGMP Signaling in Response to Nitric Oxide and Atrial Natriuretic Peptide in Vascular Smooth Muscle Cells

Cyclic 3\u27,5\u27-guanosine monophosphate (cGMP) is a key signaling molecule involved in a myriad of physiological processes, including vascular smooth muscle (VSM) tone, water- and electrolyte homeostasis, platelet aggregation, airway smooth muscle tone, smooth muscle proliferation and bone formation. Increased occurrence of vascular disorders including erectile dysfunction, hypertension, stroke and coronary artery disease, have made it increasingly important to study the dynamic interplay between cGMP synthesis and hydrolysis in VSM cells. This dissertation examines the spatial distribution of intracellular cGMP, [cGMP]i, in response to NO and atrial natriuretic peptide (ANP) in VSM cells. To investigate the spatial patterning of [cGMP]i, we have developed a new generation of non-FRET (fluorescence resonance energy transfer) cGMP biosensors that are suitable to monitor [cGMP]i in response to physiological (low-nanomolar) NO and ANP concentrations and that qualify for real-time, confocal imaging techniques. We have termed these indicators FlincGs, for green fluorescent indicators of cGMP. For the development of FlincGs, we made use of the specific cGMP binding characteristics of PKG. We utilized site-specific mutagenesis, kinetic cGMP binding, dissociation and kinase assays, as well as crystallography, in order to investigate PKG activation and cGMP binding dynamics in greater detail. Based on these studies, our novel, non-FRET cGMP biosensors were designed by attaching cGMP binding fragments of PKG to the N-terminus of circular permutated green fluorescent protein. We applied FlincGs in cultured VSM cells as well as in intact tissue to determine whether two spatially distinct populations of guanlylyl cyclase (cytosolic versus membrane bound) underlie the generation of spatiotemporally-specific patterns of [cGMP]i formation

Association between Micronutrients (Vitamin A, D, Iron) and Schistosome-Specific Cytokine Responses in Zimbabweans Exposed to Schistosoma haematobium

Micronutrients play an important role in the development of effective immune responses. This study characterised a populations exposed to schistosome infections in terms of the relationship between micronutrients and immune responses. Levels of retinol binding protein (RBP; vitamin A marker), vitamin D, ferritin and soluble transferrin receptor (sTfR), and C reactive protein (CRP) were related to levels of schistosome specific cytokines (IFN-γ, IL-4/5/10) in 40 Zimbabweans (7–54 years) exposed to Schistosoma haematobium infection. 67.2% of the participants were deficient in vitamin D. RBP levels were within normal ranges but declined with age. The two indicators of iron levels suggested that although levels of stored iron were within normal levels (normal ferritin levels), levels of functional iron (sTfR levels) were reduced in 28.6% of the population. Schistosome infection alone was not associated with levels of any of the micronutrients, but altered the relationship between parasite-specific IL-4 and IL-5 and levels of ferritin and sTfR

Der kryptogene therapierefraktäre Status epilepticus mit und ohne vorherigem Fieber bei Erwachsenen und Kindern: Eine retrospektive, monozentrische, epidemiologische Beobachtungsstudie zum Vergleich von New-Onset Refractory Status Epilepticus (NORSE) bei Erwachsenen und Febrile Infection-Related Epilepsy Syndrome (FIRES) bei Kindern

Fragestellung: Febrile Infection-Related Epilepsy Syndrom (FIRES) gilt als Untergruppe von New-Onset Refractory Status Epilepticus (NORSE). Der Zusammenhang zwischen FIRES und NORSE wird kontrovers diskutiert und es ist ungeklärt, ob es sich bei beiden um eine oder verschiedene Entitäten handelt. Um diese Frage zu klären, wurde zum ersten Mal innerhalb einer Studie eine FIRES-Kohorte mit einer NORSE-Kohorte verglichen. Methoden: Es wurden 52 Kinder mit kryptogenem FIRES und 18 Erwachsene mit kryptogenem NORSE verglichen. Da bei acht der Erwachsenen mit NORSE nicht bekannt war, ob sie einen fieberhaften Infekt während der Prodromalphase hatten, wurden drei Gruppen untersucht: 1) FIRES, 2) NORSE, 3) afebriler NORSE. Ergebnisse: Bei NORSE erkrankten häufiger Frauen (78 % bei NORSE vs. 42 % bei FIRES, p = 0,009). Die mediane akute Krankenhausaufenthaltsdauer war bei FIRES länger als bei NORSE (36 Tage [IQA = 37] vs. 20 Tage [IQA = 19]; p < 0.001). Kinder mit FIRES wurden häufiger mit Komatherapie behandelt als Erwachsene mit NORSE (81 % vs. 28 %; p < 0.001). Kinder mit FIRES erhielten häufiger Immuntherapie als Erwachsene mit NORSE (73% vs. 22 %, p < 0,001), welche bei den Kindern mit FIRES jedoch seltener einen positiven Effekt hatte (24 % bei FIRES vs. 67 % bei NORSE). Schlussfolgerung: Die Studie ergab mehr Unterschiede als Gemeinsamkeiten zwischen kryptogenem NORSE und FIRES. Es bleibt unklar, ob die Unterschiede auf das unterschiedliche Alter oder das Vorhandensein oder Fehlen von Fieber während der Prodromalphase zurückzuführen sind. Prospektiv sollten altersgetrennt NORSE- und FIRES-Kohorten untersucht werden, sodass der Einfluss des Fiebers während der Prodromalphase genauer bestimmt werden kann

Colorimetric Chemical Differentiation and Detection of Phosphorus in Eutrophic and High Particulate Waters: Advantages of a New Monitoring Approach

Phosphorus (P) is a key factor forcing eutrophication in limnic and marine systems, and all monitoring programs for water quality accordingly include P determinations. However, traditional monitoring does not allow an analysis of the different components involved in the P cycle taking place in the water column. Nonetheless, the implementation of measures addressing eutrophication requires a full understanding of the processes involved in the transformation and transport of P, in all its chemical forms. In this study, the P categories present in a river and its estuary in northern Germany, which discharge into the Baltic Sea, were characterized. Using the molybdenum blue method we found that the classification of P into the traditional fractions (DIP, DOP, POP) applied in the ocean cannot be applied to turbid waters such as rivers because interferences between the fractions seems to occur. Therefore a new nomenclature has been introduced. In addition to total phosphorus (TP) and dissolved molybdate-reactive phosphorus (DRP; previously referred to as inorganic phosphorus), dissolved non-molybdate-reactive phosphorus (DNP), particulate molybdatereactive phosphorus (PRP), and particulate non-molybdate-reactive phosphorus (PNP) were distinguished. The high spatial and temporal variations in the proportions of these forms with respect to the TP concentration well-demonstrate the complexity of the P cycle and the involved P fractions and emphasize the need for expanded monitoring approach. The potential of eutrophication could be underestimated if not all P categories were considered. With the new operational nomenclature the common and standardized molybdenum blue reaction could be used to implement the analysis of various P components into regular monitoring programs

Nutrient turnover at the sediment/water interface in shallow eutrophic coastal waters

The nutrient situation in eutrophic shallow coastal water systems is characterized by particular features. In addition to water exchange with the open sea, interactions at the sediment/water interface play a significant role. With the help of investigation on phosphate sorption/desorption balances and on the denitrification capacity of shallow coastal waters, the following general conclusions can be made: The phosphate concentrations in the water are primarily determined by physico-chemical reactions with the sediment, and less by the rhythm of the phytoplankton primary production. Through intensive interactions between sediment and water, a phosphate "equilibrium" concentration which fluctuates very little is maintained over the whole year. High primary production rates are possible at these low equilibrium concentrations of phosphate. The phosphate sorption capacity of sediments is a characteristic parameter of shallow systems. Sediments with a high organic matter content exhibit the highest phosphate sorption capacities. With the development of nearly anearobic conditions, nitrate reduction can occur at a rate, when the appropriate amount of nitrate is made available to the reaction. Under optimal conditions for nitrate reduction, nitrate concentration is also not a parameter characterizing the nature of the water body

Recombinant production of the human complement factor 5a in Escherichia coli

Up to now, the human complement factor 5a (C5a) has only been produced in small quantities in Escherichia coli in a soluble, bioactive conformation, which is not suitable for commercial production systems. This stems from the extremely high instability of C5a, as well as its aggregation-prone nature. Therefore, we analyzed several different methods for optimizing the solubility and biological activity of C5a produced by E. coli. The solubility of C5a was efficiently improved by expressing it as a glutathione-S-transferase (GST) fusion protein and, to a lesser extent, by lowering the cultivation temperature. Neither reducing the inductor concentration (isopropylthio-β-galactoside, IPTG) of the T7lac promotor nor the concomitant overexpression of endogenous chaperones was effective. However, the biological activity of the protein was improved by the overexpression of chaperones together with cultivation at 22°C, while fusion to GST slightly reduced its activity. Consequently, low cultivation temperature and the overexpression of chaperones seem to be the optimal strategy for expression of appropriate amounts of soluble and functional C5a. These findings should be the basis for the transfer to large-scale fermentation. Using C5a as an example, we showed that strain engineering in combination with specific cultivation conditions improve the production of difficult-to-express proteins in appropriate amounts and in a functional conformation facilitating the commercial manufacturing under good manufacturing practices (GMP) conditions.Keywords: Complement factor 5a (C5a), Origami 2, BL21, periplasm, cytoplasm, chaperones, Glutathione-S-Transferase (GST), temperatur

Nitrous oxide water column distribution during the transition from anoxic to oxic conditions in the Baltic Sea

International audienceIn January 2003, a major inflow of cold and oxygen-rich North Sea Water in the Baltic Sea terminated an ongoing stagnation period in parts of the central Baltic Sea. In order to investigate the role of North Sea Water inflow to the Baltic Sea with regard to the production of nitrous oxide (N2O), we measured dissolved and atmospheric N2O at 26 stations in the southern and central Baltic Sea in October 2003. At the time of our cruise, water renewal had proceeded to the eastern Gotland Basin, whereas the western Gotland Basin was still unaffected by the inflow. The deep water renewal was detectable in the distributions of temperature, salinity, and oxygen concentrations as well as in the distribution of the N2O concentrations: Shallow stations in the Kiel Bight and Pomeranian Bight were well-ventilated with uniform N2O concentrations near equilibrium throughout the water column. In contrast, stations in the deep basins, such as the Bornholm and the Gotland Deep, showed a clear stratification with deep water affected by North Sea Water. Inflowing North Sea Water led to changed environmental conditions, especially enhanced oxygen (O2) or declining hydrogen sulfide (H2S) concentrations, thus, affecting the conditions for the production of N2O. Pattern of N2O profiles and correlations with parameters like oxygen and nitrate differed between the basins. The dominant production pathway seems to be nitrification rather than denitrification. No indications for advection of N2O by North Sea Water were found. A rough budget revealed a significant surplus of in situ produced N2O after the inflow. However, due to the permanent halocline, it can be assumed that the formed N2O does not reach the atmosphere. Hydrographic aspects therefore are decisive factors determining the final release of produced N2O to the atmosphere

Distribution of N₂O in the Baltic Sea during transition from anoxic to oxic conditions

In January 2003, a major inflow of cold and oxygen-rich North Sea Water terminated an ongoing stagnation period in parts of the central Baltic Sea. In order to investigate the role of North Sea Water inflow in the production of nitrous oxide (N2O), we measured dissolved and atmospheric N<2O at 26 stations in the southern and central Baltic Sea in October 2003. At the time of our cruise, water renewal had proceeded to the eastern Gotland Basin, whereas the western Gotland Basin was still unaffected by the inflow. The deep water renewal was detectable in the distributions of temperature, salinity, and oxygen concentrations as well as in the distribution of the N2O concentrations: Shallow stations in the Kiel Bight and Pomeranian Bight were well-ventilated with uniform N2O concentrations near equilibrium throughout the water column. In contrast, stations in the deep basins, such as the Bornholm and the Gotland Deep, showed a clear stratification with deep water affected by North Sea Water. Inflowing North Sea Water led to changed environmental conditions, especially enhanced oxygen (O2) or declining hydrogen sulphide (H2S) concentrations, thus, affecting the conditions for the production of N2O. Pattern of N2O profiles and correlations with parameters like oxygen and nitrate differed between the basins. Because of the positive correlation between ΔN2O and AOU in oxic waters the dominant production pathway seems to be nitrification rather than denitrification. Advection of N2O by North Sea Water was found to be of minor importance. A rough budget revealed a significant surplus of in situ produced N2O after the inflow. However, due to the permanent halocline, it can be assumed that the N2O produced does not reach the atmosphere. Hydrographic aspects therefore are decisive factors determining the final release of N2O produced to the atmosphere