Evaluierung von Lösungswegen zur cross-medialen Umsetzung eines wissenschaftlichen Periodikums

In der vorliegenden Arbeit werden verschiedene Lösungswege zur cross-medialen Umsetzung eines wissenschaftlichen Periodikums evaluiert. Die Anforderungen werden anhand eines konkreten Anwendungsbeispiels – der Fachzeitschrift ‚inSiDE’ – formuliert. Da dieses Anwendungsbeispiel sowohl als Printausgabe als auch Online-Medium erscheint, muss der Web Workflow in den bereits vorhandenen Print Workflow integriert werden, um einen gemeinsamen Publikationsprozess zu schaffen. Der besondere Evaluationsbedarf besteht in der Erstellung, Verarbeitung und Darstellung mathematischer Ausdrücke in einem Publikationsprozess wissenschaftlicher Texte. Dieser Problematik wird folgerichtig besondere Aufmerksamkeit gewidmet

A link of Ca(2+ )to cAMP oscillations in Dictyostelium: the calmodulin antagonist W-7 potentiates cAMP relay and transiently inhibits the acidic Ca(2+)-store

BACKGROUND: During early differentiation of Dictyostelium the attractant cAMP is released periodically to induce aggregation of the cells. Here we pursue the question whether pulsatile cAMP signaling is coupled to a basic Ca(2+)-oscillation. RESULTS: We found that the calmodulin antagonist W-7 transiently enhanced cAMP spikes. We show that W-7 acts on an acidic Ca(2+)-store: it abolished ATP-dependent vesicular acidification, inhibited V-type H(+)ATPase activity more potently than the weaker antagonist W-5 and caused vesicular Ca(2+)-leakage. Concanamycin A, an inhibitor of the V-type H(+)-pump, blocked the Ca(2+)-leakage elicited by W-7 as well as cAMP-oscillations in the presence of W-7. Concanamycin A caused an increase of the cytosolic Ca(2+)-concentration whereas W-7 did not. In case of the latter, Ca(2+ )was secreted by the cells. In accord with our hypothesis that the link from Ca(2+ )to cAMP synthesis is mediated by a Ca(2+)-dependent phospholipase C we found that W-7 was not active in the phospholipase C knockout mutant. CONCLUSION: We conclude that the potentiation of cAMP relay by W-7 is due to a transient inhibition of the acidic Ca(2+)-store. The inhibition of the proton pump by W-7 causes a leakage of Ca(2+ )that indirectly stimulates adenylyl cyclase activity via phospholipase C

Funktionalisierte Nukleinsäuren : Studien & Selektion von Ribozymen

In der vorliegenden Arbeit wurden die für die in vitro Selektion katalytisch aktiver Nukleinsäuren erforderlichen Techniken weiterentwickelt und die Einsetzbarkeit von Ribozymen in der organischen Chemie unter qualitativen und quantitativen Gesichtspunkten evaluiert. Für die in vitro Selektion neuer RNA/DNA-Katalysatoren ist generell die Derivatisierbarkeit von Nukleinsäuren essentiell. Je mehr Möglichkeiten bestehen, in Oligonukleotide funktionelle Gruppen einzuführen, desto größer ist die mögliche Diversität zu isolierender Katalysatoren. Innerhalb dieser Doktorarbeit konnten generell zwei unterschiedliche Funktionalisierungsansätze erarbeitet werden. Über enzymatische Reaktionen konnten sowohl an terminalen wie auch an molekülinternen Positionen von Nukleinsäuren Funktionalitäten eingefügt werden. Die Synthese zweier Initiatornukleotide zum terminalen Einbau einer Thiol- bzw. Amino-Funktionalisierung in Ribonukleinsäuren ist ein Ergebnis dieser Arbeit. Die jeweilige Struktur der Initiatornukleotide konnte mittels MALDI-MS gesichert, ihr Einbau durch T7-RNA-Polymerase in Oligonukleotide per Autoradiographie von Polyacrylamidgelen gezeigt und durch anschließende Umsetzung mit Biotinmaleimid (Initiator-SH-nukleotid) bzw. Sulfo-NHS-Biotin (Initiator-NH2-nukleotid) ihre Derivatisierbarkeit bewiesen werden. Ferner konnte gezeigt werden, dass die molekülinterne Funktionalisierung von Desoxyribonukleinsäuren durch Verwendung einer sequenzspezifischen Methyltransferase (M.TaqI) und eines biotinylierten Cofaktoranalogons möglich ist. Die intern funktionalisierte DNA konnte mittels PCR amplifiziert werden. Dadurch wurde die Kompatibilität mit einem aufgestellten Selektionsschema bewiesen. Die Bildung von Kohlenstoff-Kohlenstoff-Bindungen wird als essentieller Bestandteil im Metabolismus einer hypothetischen RNA-Welt angesehen. Künstliche RNA-Katalysatoren wie das in unserer Arbeitsgruppe entwickelte Diels-Alderase-Ribozym können diese These stützen und darüber hinaus Ausgangspunkt für den Einsatz in vitro selektierter Biokatalysatoren in der Wirkstoffsynthese darstellen. Die praktische Verwendung solcher maßgeschneiderter Ribozyme in der Synthese hängt von den Produktionskosten, Stabilität und Effektivität des Katalysators ab. In Hinblick auf die zukünftige Wirtschaftlichkeit ribozymkatalysierter Reaktionen wurde hier partiell die Technologiebasis für einen Ribozymreaktor geschaffen. Das 49-mer-Minimalmotiv des Diels-Alderase Ribozyms wurde nahezu quantitativ kovalent an einer Festphase immobilisiert. Die katalytische Aktivität und Selektivität blieben dabei erhalten. Die aktivierte Festphase wies eine sehr gute Langzeit-Stabilität auf. Dadurch wurden wesentliche Kriterien bezüglich der Konstruktion eines Ribozymreaktors zur ökonomischen Synthese erfüllt. Zur Umsetzung dieser Idee muss die Effektivität des immobilisierten Ribozyms gesteigert werden. Um dieses Ziel zu erreichen wurden neue, effektivere Diels-Alderasen generiert und charakterisiert. Dabei wurden katalytische RNAs aus einer angereicherten, kombinatorischen RNA-Bibliothek mit Hilfe der in vitro Selektionstechnik durch Einsatz einer Quenched-Flow-Apparatur isoliert. Der Selektionsdruck wurde durch das Herabsenken der Reaktionszeit bis in den Millisekundenbereich erhöht. Nach 17 Selektionsrunden wurde die Selektion beendet. 19 Diels-Alderase-Ribozyme konnten nach Klonierung und Sequenzierung mittels fluoreszenzspektromtetrischen Assays kinetisch charakterisiert werden. Demnach beschleunigen die aktivsten Sequenzen die Reaktion zwischen Anthracen-Konjugat und Biotinmaleimid um das ca. 72000fache und weisen eine apparente Geschwindigkeitskonstante von ca. 940 M-1s-1 auf. Die aktivsten Sequenzen der Selektion beschleunigen die Diels-Alder-Reaktion 3.5 mal besser als das bislang immobilisierten 49-mer des Diels-Alderase-Ribozyms in vergleichbaren Studien

cAMP controls cytosolic Ca(2+ )levels in Dictyostelium discoideum

BACKGROUND: Differentiating Dictyostelium discoideum amoebae respond upon cAMP-stimulation with an increase in the cytosolic free Ca(2+ )concentration ([Ca(2+)](i)) that is composed of liberation of stored Ca(2+ )and extracellular Ca(2+)-influx. In this study we investigated whether intracellular cAMP is involved in the control of [Ca(2+)](i). RESULTS: We analyzed Ca(2+)-fluxes in a mutant that is devoid of the main cAMP-phosphodiesterase (PDE) RegA and displays an altered cAMP metabolism. In suspensions of developing cells cAMP-activated influx of extracellular Ca(2+ )was reduced as compared to wild type. Yet, single cell [Ca(2+)](i)-imaging of regA(- )amoebae revealed a cAMP-induced [Ca(2+)](i )increase even in the absence of extracellular Ca(2+). The cytosolic presence of the cAMP PDE inhibitor 3-isobutyl-1-methylxanthine (IBMX) induced elevated basal [Ca(2+)](i )in both, mutant and wild type cells. Under this condition wild type cells displayed cAMP-activated [Ca(2+)](i)-transients also in nominally Ca(2+)-free medium. In the mutant strain the amplitude of light scattering oscillations and of accompanying cAMP oscillations were strongly reduced to almost basal levels. In addition, chemotactic performance during challenge with a cAMP-filled glass capillary was altered by EGTA-incubation. Cells were more sensitive to EGTA treatment than wild type: already at 2 mM EGTA only small pseudopods were extended and chemotactic speed was reduced. CONCLUSION: We conclude that there is a link between the second messengers cAMP and Ca(2+). cAMP-dependent protein kinase (PKA) could provide for this link as a membrane-permeable PKA-activator also increased basal [Ca(2+)](i )of regA(- )cells. Intracellular cAMP levels control [Ca(2+)](i )by regulating Ca(2+)-fluxes of stores which in turn affect Ca(2+)-influx, light scattering oscillations and chemotactic performance

The contractile vacuole in Ca(2+)-regulation in Dictyostelium: its essential function for cAMP-induced Ca(2+)-influx

BACKGROUND: cAMP-induced Ca(2+)-influx in Dictyostelium is controlled by at least two non-mitochondrial Ca(2+)-stores: acidic stores and the endoplasmic reticulum (ER). The acidic stores may comprise the contractile vacuole network (CV), the endosomal compartment and acidocalcisomes. Here the role of CV in respect to function as a potential Ca(2+)-store was investigated. RESULTS: Dajumin-GFP labeled contractile vacuoles were purified 7-fold by anti-GFP-antibodies in a magnetic field. The purified CV were shown for the first time to accumulate and release Ca(2+). Release of Ca(2+ )was elicited by arachidonic acid or the calmodulin antagonist W7, the latter due to inhibition of the pump. The characteristics of Ca(2+)-transport and Ca(2+)-release of CV were compared to similarly purified vesicles of the ER labeled by calnexin-GFP. Since the CV proved to be a highly efficient Ca(2+)-compartment we wanted to know whether or not it takes part in cAMP-induced Ca(2+)-influx. We made use of the LvsA(-)-mutant expected to display reduced Ca(2+)-transport due to loss of calmodulin. We found a severe reduction of cAMP-induced Ca(2+)-influx into whole cells. CONCLUSION: The contractile vacuoles in Dictyostelium represent a highly efficient acidic Ca(2+)-store that is required for cAMP-induced Ca(2+)-influx

Ca(2+ )regulation in the absence of the iplA gene product in Dictyostelium discoideum

BACKGROUND: Stimulation of Dictyostelium discoideum with cAMP evokes an elevation of the cytosolic free Ca(2+ )concentration ([Ca(2+)](i)). The [Ca(2+)](i)-change is composed of liberation of stored Ca(2+ )and extracellular Ca(2+)-entry. The significance of the [Ca(2+)](i)-transient for chemotaxis is under debate. Abolition of chemotactic orientation and migration by Ca(2+)-buffers in the cytosol indicates that a [Ca(2+)](i)-increase is required for chemotaxis. Yet, the iplA(- )mutant disrupted in a gene bearing similarity to IP(3)-receptors of higher eukaryotes aggregates despite the absence of a cAMP-induced [Ca(2+)](i)-transient which favours the view that [Ca(2+)](i)-changes are insignificant for chemotaxis. RESULTS: We investigated Ca(2+)-fluxes and the effect of their disturbance on chemotaxis and development of iplA(- )cells. Differentiation was altered as compared to wild type amoebae and sensitive towards manipulation of the level of stored Ca(2+). Chemotaxis was impaired when [Ca(2+)](i)-transients were suppressed by the presence of a Ca(2+)-chelator in the cytosol of the cells. Analysis of ion fluxes revealed that capacitative Ca(2+)-entry was fully operative in the mutant. In suspensions of intact and permeabilized cells cAMP elicited extracellular Ca(2+)-influx and liberation of stored Ca(2+), respectively, yet to a lesser extent than in wild type. In suspensions of partially purified storage vesicles ATP-induced Ca(2+)-uptake and Ca(2+)-release activated by fatty acids or Ca(2+)-ATPase inhibitors were similar to wild type. Mn(2+)-quenching of fura2 fluorescence allows to study Ca(2+)-influx indirectly and revealed that the responsiveness of mutant cells was shifted to higher concentrations: roughly 100 times more Mn(2+ )was necessary to observe agonist-induced Mn(2+)-influx. cAMP evoked a [Ca(2+)](i)-elevation when stores were strongly loaded with Ca(2+), again with a similar shift in sensitivity in the mutant. In addition, basal [Ca(2+)](i )was significantly lower in iplA(- )than in wild type amoebae. CONCLUSION: These results support the view that [Ca(2+)](i)-transients are essential for chemotaxis and differentiation. Moreover, capacitative and agonist-activated ion fluxes are regulated by separate pathways that are mediated either by two types of channels in the plasma membrane or by distinct mechanisms coupling Ca(2+)-release from stores to Ca(2+)-entry in Dictyostelium. The iplA(- )strain retains the capacitative Ca(2+)-entry pathway and an impaired agonist-activated pathway that operates with reduced efficiency or at higher ionic pressure

Plasma responses in human subjects after ingestions of multiple doses of natural α-cryptoxanthin: A pilot study

Xanthophylls have attracted a lot of interest since their health benefits were documented. Unfortunately, studying their intestinal absorption is often affected by high baseline levels present in the fasting plasma. As a-cryptoxanthin is rarely found in the traditional European diet, its concentration in human plasma is extremely low. A pilot human intervention study was designed using a-cryptoxanthin for the first time as a marker xanthophyll in a minimally formulated cellulose-based supplement. α-Cryptoxanthin was administered in gelatin soft-gel capsules in multiple doses of 156 μg/d to three male volunteers (age 27.3 (SD 4.7) years; BMI 21.6 (SD 0.3) kg/m2) for 16 d after a 2-week carotenoid depletion period. Fasting blood samples were taken before the intervention and after 3, 6, 9, 13 and 16 d. Plasma HPLC analyses allowed for determination of the concentration; liquid chromatography-MS in the single ion monitoring mode was used to confirm peak assignment. The concentrations of α-cryptoxanthin increased significantly after only 3 d of supplementation. The concentration-time plots showed a characteristic shape with a first maximum after day 6, a decline until day 9 and a gradual second rise until the end of the study. standardisation of plasma α-cryptoxanthin concentrations to triacylglycerol or total cholesterol did not influence the characteristics. The maximum concentrations reached at the end of the intervention period ranged from 0.077 to 0.160 μmol/l. These results suggest a high intestinal absorption and an enrichment of α-cryptoxanthin in the plasma even from a minimally formulated cellulose-based supplement. © The Authors 2006

Assessment of heat generation and its effect during cortical bone drilling using infrared camera and histology

182-188Orthopedic bone drilling involves human as a part of action thus the role of drilling must be concise with its prime objective only. Excess heat generation and physical damage during drilling will lead to extended problems i.e. osteonecrosis and permanent death to the bone cells. To avoid that particular lose to the bone the heat generation should be as low as possible. In this study, an experiment is performed on bovine bone with varying rotational speed (600, 800, 1000, 1200 RPM) while keeping all other drilling parameters constant. Heat generation during bone drilling is measured using thermal imaging camera. After experiments, the histology examinations are performed to observe by morphological changes in drilled bones. From results, heat generation is observed to be increased with the rotational speed and results are shown with the help of thermo-graphic images. Histopathology of drilled bone specimens is also carried out for better understanding of changes in morphology of bone with change in temperature raise during bone drilling. Results conclude that heat generation in bone drilling is strongly concord with drill rotational speed (P≤0.014). Histopathology of drilled bones shows that level of osteonecrosis is increased in terms of number of empty lacunas with temperature raise

Assessment of heat generation and its effect during cortical bone drilling using infrared camera and histology

Orthopedic bone drilling involves human as a part of action thus the role of drilling must be concise with its prime objective only. Excess heat generation and physical damage during drilling will lead to extended problems i.e. osteonecrosis and permanent death to the bone cells. To avoid that particular lose to the bone the heat generation should be as low as possible. In this study, an experiment is performed on bovine bone with varying rotational speed (600, 800, 1000, 1200 RPM) while keeping all other drilling parameters constant. Heat generation during bone drilling is measured using thermal imaging camera. After experiments, the histology examinations are performed to observe by morphological changes in drilled bones. From results, heat generation is observed to be increased with the rotational speed and results are shown with the help of thermo-graphic images. Histopathology of drilled bone specimens is also carried out for better understanding of changes in morphology of bone with change in temperature raise during bone drilling. Results conclude that heat generation in bone drilling is strongly concord with drill rotational speed (P≤0.014). Histopathology of drilled bones shows that level of osteonecrosis is increased in terms of number of empty lacunas with temperature raise