Miniemulsionen als räumliche Begrenzungen : Synthese von Polymer-Hydroxylapatit-Nanopartikeln, bio-inspirierten Nanokapseln und Polymer-Einzelkettenpartikeln

In der vorliegenden Arbeit wurden Miniemulsionen als räumliche Begrenzungen für die Synthese von unterschiedlichen funktionellen Materialien mit neuartigen Eigenschaften verwendet. Das erste Themengebiet umfasst die Herstellung von Polymer/Calciumphosphat-Hybridpartikeln und –Hybridkapseln über die templatgesteuerte Mineralisation von Calciumphosphat. Die funktionalisierte Oberfläche von Polymernanopartikeln, welche über die Miniemulsionspolymerisation hergestellt wurden, diente als Templat für die Kristallisation von Calciumphosphat auf den Partikeln. Der Einfluss der funktionellen Carboxylat- und Phosphonat-Oberflächengruppen auf die Komplexierung von Calcium-Ionen sowie die Mineralisation von Calciumphosphat auf der Oberfläche der Nanopartikel wurde mit mehreren Methoden (ionenselektive Elektroden, REM, TEM und XRD) detailliert analysiert. Es wurde herausgefunden, dass die Mineralisation bei verschiedenen pH-Werten zu vollkommen unterschiedlichen Kristallmorphologien (nadel- und plättchenförmige Kristalle) auf der Oberfläche der Partikel führt. Untersuchungen der Mineralisationskinetik zeigten, dass die Morphologie der Hydroxylapatit-Kristalle auf der Partikeloberfläche mit der Änderung der Kristallisationsgeschwindigkeit durch eine sorgfältige Wahl des pH-Wertes gezielt kontrolliert werden kann. Sowohl die Eigenschaften der als Templat verwendeten Polymernanopartikel (z. B. Größe, Form und Funktionalisierung), als auch die Oberflächentopografie der entstandenen Polymer/Calciumphosphat-Hybridpartikel wurden gezielt verändert, um die Eigenschaften der erhaltenen Kompositmaterialien zu steuern. rnEine ähnliche bio-inspirierte Methode wurde zur in situ-Herstellung von organisch/anorganischen Nanokapseln entwickelt. Hierbei wurde die flexible Grenzfläche von flüssigen Miniemulsionströpfchen zur Mineralisation von Calciumphosphat an der Grenzfläche eingesetzt, um Gelatine/Calciumphosphat-Hybridkapseln mit flüssigem Kern herzustellen. Der flüssige Kern der Nanokapseln ermöglicht dabei die Verkapselung unterschiedlicher hydrophiler Substanzen, was in dieser Arbeit durch die erfolgreiche Verkapselung sehr kleiner Hydroxylapatit-Kristalle sowie eines Fluoreszenzfarbstoffes (Rhodamin 6G) demonstriert wurde. Aufgrund der intrinsischen Eigenschaften der Gelatine/Calciumphosphat-Kapseln konnten abhängig vom pH-Wert der Umgebung unterschiedliche Mengen des verkapselten Fluoreszenzfarbstoffes aus den Kapseln freigesetzt werden. Eine mögliche Anwendung der Polymer/Calciumphosphat-Partikel und –Kapseln ist die Implantatbeschichtung, wobei diese als Bindeglied zwischen künstlichem Implantat und natürlichem Knochengewebe dienen. rnIm zweiten Themengebiet dieser Arbeit wurde die Grenzfläche von Nanometer-großen Miniemulsionströpfchen eingesetzt, um einzelne in der dispersen Phase gelöste Polymerketten zu separieren. Nach der Verdampfung des in den Tröpfchen vorhandenen Lösungsmittels wurden stabile Dispersionen sehr kleiner Polymer-Nanopartikel (<10 nm Durchmesser) erhalten, die aus nur wenigen oder einer einzigen Polymerkette bestehen. Die kolloidale Stabilität der Partikel nach der Synthese, gewährleistet durch die Anwesenheit von SDS in der wässrigen Phase der Dispersionen, ist vorteilhaft für die anschließende Charakterisierung der Polymer-Nanopartikel. Die Partikelgröße der Nanopartikel wurde mittels DLS und TEM bestimmt und mit Hilfe der Dichte und des Molekulargewichts der verwendeten Polymere die Anzahl an Polymerketten pro Partikel bestimmt. Wie es für Partikel, die aus nur einer Polymerkette bestehen, erwartet wird, stieg die mittels DLS bestimmte Partikelgröße mit steigendem Molekulargewicht des in der Synthese der Partikel eingesetzten Polymers deutlich an. Die Quantifizierung der Kettenzahl pro Partikel mit Hilfe von Fluoreszenzanisotropie-Messungen ergab, dass Polymer-Einzelkettenpartikel hoher Einheitlichkeit hergestellt wurden. Durch die Verwendung eines Hochdruckhomogenisators zur Herstellung der Einzelkettendispersionen war es möglich, größere Mengen der Einzelkettenpartikel herzustellen, deren Materialeigenschaften zurzeit näher untersucht werden.rn

Bioverkapselung lebender Bakterien (Escherichia coli) mit Poly-(Silicat) nach Transformation mit dem Silicatein-alpha-Gen

Die Bioverkapselung ist eine faszinierende Methode, um biologische Materialien einschließlich Zellen in Siliziumdioxid, Metalloxiden oder hybriden Sol-Gel-Polymeren zu immobilisieren. Bisher wurde nur die Sol-Gel-Vorläufertechnologie genutzt, um Bakterien- oder Hefezellen in Siliziumdioxid zu immobilisieren. Hierfür wurden verschiedene Reagenzien als wässrige Vorläufer getestet, um poly(Silicate) auf Biomolekülen (Bhatia et al., 2000) oder Zellen (Liu und Chen 1999; Coradin und Livage, 2007) zu bilden. Einer der erfolgreichsten bisherigen Methoden verwendet eine Mischung aus Silicaten und kolloidalem Silica. Diese initialen Vorläufer werden durch die Zugabe von Salzsäure neutralisiert, was die Gelbildung fortschreiten lässt und die Verkapselung von Bakterien in einem Silica-Netzwerk zur Folge hat (Nassif et al., 2003). Mit der Entdeckung von Silicatein, einem Enzym, das aus Demospongien isoliert wurde und die Bildung von poly(Silicat) katalysiert, wurde es möglich, poly(Silicat) unter physiologischen Bedingungen zu synthetisieren. Silicatein wurde rekombinant in E. coli hergestellt und ist in der Lage, bei Raumtemperatur, neutralem pH-Wert und in wässrigen Puffersystemen aus Siliziumalkoxiden poly(Silicat) zu bilden (Krasko et al., 2000; Müller et al., 2007b; Zhou et al., 1999). In vivo katalysiert Silicatein die Synthese der Silicathülle der Schwamm-Spiculae (Skelettelemente; Müller et al., 2005b; Müller et al., 2007a; Müller et al., 2007b; Schröder et al., 2007a). Dieses Biosilica wurde in Form von Silica-Nanospheren mit Durchmessern zwischen 100 nm und 250 nm organisiert vorgefunden (Pisera 2003; Tahir et al., 2005). Mit dieser Arbeit konnte gezeigt werden, dass Escherichia coli erfolgreich mit dem Silicatein-Gen transformiert werden kann. Das Level der Proteinexpression kann in Anwesenheit von Isopropyl-β-D-thiogalaktopyranosid (IPTG) effizient erhöht werden, indem man die Bakterienzellen gleichzeitig mit Kieselsäure inkubiert. Dieser Effekt konnte sowohl auf Ebene der Synthese des rekombinanten Proteins durch Western Blot als auch durch Immunfluoreszenzmikroskopie nachgewiesen werden. Das heterolog produzierte Silicatein besitzt enzymatische Aktivität und kann die Polymerisation von Kieselsäure katalysieren. Dies konnte sowohl durch Färbung mit Rhodamin123, als auch durch Reaktion der nicht polymerisierten, freien Kieselsäure mit dem ß-Silicomolybdato-Farbsystem (Silicomolybdänblau) nachgewiesen werden. Elektronenmikroskopische Untersuchungen zeigten, dass nur die silicateinexprimierenden Bakterien während des Wachstums in Anwesenheit von Kieselsäure eine viskose Hülle um Zelle herum bilden. Ebenfalls konnte gezeigt werden, dass Silicatein-α aus Suberites domuncula nach Transformation in E. coli an die Zelloberfläche dieser Zellen transportiert wurde und dort seine enzymatische Funktion beibehielt. Die Silicathülle wurde mittels Raster-Elektronenmikroskopie (REM) analysiert. Die Bakterien, die Silicatein exprimierten und poly(Silicat) an ihrer Oberfläche synthetisierten, zeigten die gleichen Wachstumsraten wie die Bakterien, die das Gen nicht enthielten. Schlussfolgernd lässt sich sagen, dass die silicateinvermittelte Verkapselung von Bakterien mit poly(Silicat) die Bandbreite der Anwendung von Bakterien für die Produktion von rekombinanten Proteinen verbessern, erweitern und optimieren könnte.

Systemic and hepatic vein galectin-3 are increased in patients with alcoholic liver cirrhosis and negatively correlate with liver function

Recently we demonstrated higher galectin-3 in portal venous serum (PVS) compared to hepatic venous serum (HVS) in a small cohort of patients with normal liver function suggesting hepatic removal of galectin-3. Here, galectin-3 was measured by ELISA in PVS, HVS and systemic venous blood (SVS) of 33 patients with alcoholic liver cirrhosis and a larger cohort of 11 patients with normal liver function. Galectin-3 was cleared by the healthy but not the cirrhotic liver, and subsequently HVS and SVS galectin-3 levels were significantly increased in the patients with liver cirrhosis compared to controls. In healthy liver galectin-3 was produced by cholangiocytes and synthesis by hepatocytes was only observed in cirrhotic liver. Hepatic venous pressure gradient did not correlate with galectin-3 levels excluding hepatic shunting as the principal cause of higher SVS galectin-3. Galectin-3 was elevated in all blood compartments of patients with CHILD-PUGH stage C compared to patients with CHILD-PUGH stage A, and was higher in patients with ascites than patients without this complication. Galectin-3 was negatively associated with antithrombin-3 whose synthesis is reduced with worse liver function. Galectin-3 positively correlated with urea and creatinine, and PVS galectin-3 showed a negative association with creatinine clearance as an accepted measure of kidney function. To summarize in the current study systemic, portal and hepatic levels of galectin-3 were found to be negatively associated with liver function in patients with alcoholic liver cirrhosis and this may in part be related to impaired hepatic removal and/or increased synthesis in cirrhotic liver.

Stable N isotope composition of nitrate reflects N transformations during the passage of water through a montane rain forest in Ecuador

Knowledge of the fate of deposited N in the possibly N-limited, highly biodiverse north Andean forests is important because of the possible effects of N inputs on plant performance and species composition. We analyzed concentrations and fluxes of NO3 −–N, NH4 +–N and dissolved organic N (DON) in rainfall, throughfall, litter leachate, mineral soil solutions (0.15–0.30 m depths) and stream water in a montane forest in Ecuador during four consecutive quarters and used the natural 15N abundance in NO3 − during the passage of rain water through the ecosystem and bulk δ15N values in soil to detect N transformations. Depletion of 15N in NO3 − and increased NO3 −–N fluxes during the passage through the canopy and the organic layer indicated nitrification in these compartments. During leaching from the organic layer to mineral soil and stream, NO3 − concentrations progressively decreased and were enriched in 15N but did not reach the δ15N values of solid phase organic matter (δ15N = 5.6–6.7‰). This suggested a combination of nitrification and denitrification in mineral soil. In the wettest quarter, the δ15N value of NO3 − in litter leachate was smaller (δ15N = −1.58‰) than in the other quarters (δ15N = −9.38 ± SE 0.46‰) probably because of reduced mineralization and associated fractionation against 15N. Nitrogen isotope fractionation of NO3 − between litter leachate and stream water was smaller in the wettest period than in the other periods probably because of a higher rate of denitrification and continuous dilution by isotopically lighter NO3 −–N from throughfall and nitrification in the organic layer during the wettest period. The stable N isotope composition of NO3 − gave valuable indications of N transformations during the passage of water through the forest ecosystem from rainfall to the stream.

The glycine deportation system and its pharmacological consequences

The glycine deportation system is an essential component of glycine catabolism in man whereby 400 to 800mg glycine per day are deported into urine as hippuric acid. The molecular escort for this deportation is benzoic acid, which derives from the diet and from gut microbiota metabolism of dietary precursors. Three components of this system, involving hepatic and renal metabolism, and renal active tubular secretion help regulate systemic and central nervous system levels of glycine. When glycine levels are pathologically high, as in congenital nonketotic hyperglycinemia, the glycine deportation system can be upregulated with pharmacological doses of benzoic acid to assist in normalization of glycine homeostasis. In congenital urea cycle enzymopathies, similar activation of the glycine deportation system with benzoic acid is useful for the excretion of excess nitrogen in the form of glycine. Drugs which can substitute for benzoic acid as substrates for the glycine deportation system have adverse reactions that may involve perturbations of glycine homeostasis. The cancer chemotherapeutic agent ifosfamide has an unacceptably high incidence of encephalopathy. This would appear to arise as a result of the production of toxic aldehyde metabolites which deplete ATP production and sequester NADH in the mitochondrial matrix, thereby inhibiting the glycine deportation system and causing de novo glycine synthesis by the glycine cleavage system. We hypothesize that this would result in hyperglycinemia and encephalopathy. This understanding may lead to novel prophylactic strategies for ifosfamide encephalopathy. Thus, the glycine deportation system plays multiple key roles in physiological and neurotoxicological processes involving glycine.

"Self" and "nonself" manipulation of interferon defense during persistent infection: bovine viral diarrhea virus resists alpha/beta interferon without blocking antiviral activity against unrelated viruses replicating in its host cells

Bovine viral diarrhea virus (BVDV), together with Classical swine fever virus (CSFV) and Border disease virus (BDV) of sheep, belongs to the genus Pestivirus of the Flaviviridae. BVDV is either cytopathic (cp) or noncytopathic (ncp), as defined by its effect on cultured cells. Infection of pregnant animals with the ncp biotype may lead to the birth of persistently infected calves that are immunotolerant to the infecting viral strain. In addition to evading the adaptive immune system, BVDV evades key mechanisms of innate immunity. Previously, we showed that ncp BVDV inhibits the induction of apoptosis and alpha/beta interferon (IFN-alpha/beta) synthesis by double-stranded RNA (dsRNA). Here, we report that (i) both ncp and cp BVDV block the induction by dsRNA of the Mx protein (which can also be induced in the absence of IFN signaling); (ii) neither biotype blocks the activity of IFN; and (iii) once infection is established, BVDV is largely resistant to the activity of IFN-alpha/beta but (iv) does not interfere with the establishment of an antiviral state induced by IFN-alpha/beta against unrelated viruses. The results of our study suggest that, in persistent infection, BVDV is able to evade a central element of innate immunity directed against itself without generally compromising its activity against unrelated viruses ("nonself") that may replicate in cells infected with ncp BVDV. This highly selective "self" and "nonself" model of evasion of the interferon defense system may be a key element in the success of persistent infection in addition to immunotolerance initiated by the early time point of fetal infection.

[Experiences of patients and family members during the stay on a nursing unit]

In 2004, the university hospital of Berne ran a pilot project with a Nursing Unit (NU). In this unit patients who no longer needed a close surveillance by physicians were cared for. They needed primarily complex professional nursing care which could not be provided by other hospitals, nursing homes, home care or family members. The nurses were responsible for the coordination of care. This qualitative study investigated experiences of patients and family members with the care concept of the NU. Thematically focused interviews were conducted with nine patients and five family members. Qualitative content analysis was used for data analysis. Results show that patients and family members mostly accepted the new care concept. They positively experienced the quiet and restful atmosphere, the patient-centred and continuous care by competent nurses, the education and the discharge planning. Some study participants reported missing information at the time of their transfer to the NU, insufficient assessments or unsuitable educational scripts. The study provides evidence to positive effects of a patient-centred care approach.

DEVELOPMENT OF THE ECOCAR 3 PROPOSAL AND GUIDELINES FOR MODELING AND DESIGN IN YEAR ONE OF ECOCAR 3

This report summarizes the work done for the Vehicle Powertrain Modeling and Design Problem Proposal portion of the EcoCAR3 proposal as specified in the Request for Proposal from Argonne National Laboratory. The results of the modeling exercises presented in the proposal showed that: An average conventional vehicle powered by a combustion engine could not meet the energy consumption target when the engine was sized to meet the acceleration target, due the relatively low thermal efficiency of the spark ignition engine. A battery electric vehicle could not meet the required range target of 320 km while keeping the vehicle weight below the gross vehicle weight rating of 2000 kg. This was due to the low energy density of the batteries which necessitated a large, and heavy, battery pack to provide enough energy to meet the range target. A series hybrid electric vehicle has the potential to meet the acceleration and energy consumption parameters when the components are optimally sized. A parallel hybrid electric vehicle has less energy conversion losses than a series hybrid electric vehicle which results in greater overall efficiency, lower energy consumption, and less emissions. For EcoCAR3, Michigan Tech proposes to develop a plug-in parallel hybrid vehicle (PPHEV) powered by a small Diesel engine operating on B20 Bio-Diesel fuel. This architecture was chosen over other options due to its compact design, lower cost, and its ability to provide performance levels and energy efficiency that meet or exceed the design targets. While this powertrain configuration requires a more complex control system and strategy than others, the student engineering team at Michigan Tech has significant recent experience with this architecture and has confidence that it will perform well in the events planned for the EcoCAR3 competition.

A new approach to generate a safe double-attenuated Plasmodium liver stage vaccine

Recently it has been shown in rodent malaria models that immunisation with genetically attenuated Plasmodium parasites can confer sterile protection against challenge with virulent parasites. For the mass production of live attenuated Plasmodium parasites for vaccination, safety is a prerequisite. Knockout of a single gene is not sufficient for such a strategy since the parasite can likely compensate for such a genetic modification and a single surviving parasite is sufficient to kill an immunised individual. Parasites must therefore be at least double-attenuated when generating a safe vaccine strain. Genetic double-attenuation can be achieved by knocking out two essential genes or by combining a single gene knockout with the expression of a protein toxic for the parasite. We generated a double-attenuated Plasmodium berghei strain that is deficient in fatty acid synthesis by the knockout of the pdh-e1α gene, introducing a second attenuation by the liver stage-specific expression of the pore-forming bacterial toxin perfringolysin O. With this double genetically attenuated parasite strain, a superior attenuation was indeed achieved compared with single-attenuated strains that were either deficient in pyruvate dehydrogenase (PDH)-E1 or expressed perfringolysin O. In vivo, both single-attenuated strains resulted in breakthrough infections even if low to moderate doses of sporozoites (2,000-5,000) were administered. In contrast, the double genetically attenuated parasite strain, given at moderate doses of 5,000 sporozoites, did not result in blood stage infection and even when administered at 5- to 20-fold higher doses, only single and delayed breakthrough infections were observed. Prime booster immunisation with the double genetically attenuated parasite strain completely protected a susceptible mouse strain from malaria and even a single immunisation conferred protection in some cases and lead to a markedly delayed onset of blood stage infection in others. Importantly, premature rupture of the parasitophorous vacuole membrane by liver stage-specific perfringolysin O expression did not induce host cell death and soluble parasite proteins, which are released into the host cell cytoplasm, have the potential to be processed and presented via MHC class I molecules. This, in turn, might support immunological responses against Plasmodium-infected hepatocytes.

The cellular and molecular involvement of glutathione in cisplatin-induced apoptosis

The goal of this study was to investigate the cellular and molecular mechanisms by which glutathione (GSH) is involved in the process of apoptosis induced by cisplatin [cis-diamminedichloroplatinum(II), cis-DDP] in the HL60 human promyelocytic leukemia cell line. The data show that during the onset or induction of apoptosis, GSH levels in cisplatin-treated cells increased 50% compared to control cells. The increase in intracellular GSH was associated with enhanced expression of γ-glutamylcysteine synthetase (γ-GCS), the enzyme that catalyzes the rate- limiting step in the biosynthesis of glutathione. After depletion of intracellular GSH with D,L-buthionine-(S,R)-sulfoximine (BSO), an inhibitor of γ-GCS, biochemical and morphological analysis revealed that the mechanism of cell death had switched from apoptosis to necrosis. In contrast, when intracellular GSH was elevated by exposure of cells to a GSH-ethyl-ester and then treatment with cisplatin, no change in the induction and kinetics of apoptosis were observed. However, when cells were exposed to cisplatin before intracellular GSH levels were increased, apoptosis was observed to occur 6 hours earlier compared to cells without GSH elevation. To further examine the molecular aspects of these effects of GSH on the apoptotic process, changes in the expression of bcl-2 and bax, were investigated in cells with depleted and elevated GSH. Using reverse transcription polymerase chain reaction, no significant change in the expression of bcl-2 gene transcripts was observed in cells in either the GSH depleted or elevated state; however, a 75% reduction in GSH resulted in a 40% decrease in the expression of bax gene transcripts. In contrast, a 6-fold increase in GSH increased the expression of bax by 3-fold relative to controls. Similar results were obtained for bax gene expression and protein synthesis by northern analysis and immunoprecipitation, respectively. These results suggest that GSH serves a dual role in the apoptotic process. The first role which is indirect, involves the protection of the cell from extensive damage following exposure to a specific toxicant so as to prevent death by necrosis, possibly by interacting with the DNA damaging agent and/or its active metabolites. The second role involves a direct involvement of GSH in the apoptotic process that includes upregulation of bax expression. ^

IMMUNOGLOBULINS IN PERIODONTAL DISEASE

Periodontal disease is the major cause of tooth loss in man. The initial histological picture of the inflamed gingiva is characteristic of local inflammatory reaction involving polymorphonuclear leukocytes, vasculitis and localized tissue loss. Subsequent clinical stages of periodontal disease (mild gingivitis) show histological evidence of the involvement of the immune response with initial accumulation of macrophages, and lymphocytes devoid of surface staining immunoglobulins (presumably T cells). As the disease progresses, a predominance of surface and cytoplasmic staining lymphocytes and plasma cells are seen (severe gingivitis and periodontitis). Whether the occurrence of the immunoglobulin positive lymphocytes and the concurrent loss of collagen and resorption of alveolar bone seen in periodontitis is indicative of a direct cause and effect relationship has been a controversy.^ The majority of investigations in the periodontal field have involved the use of peripheral blood lymphocytes or serum. Blastogenic responses of peripheral blood lymphocytes and serum antibody titers from periodontal patients to a variety of oral bacteria have not shown any correlation between response and the severity of disease. The need to study the local immune response in inflamed gingiva is apparent. Since there are no baseline studies on the functional capabilities of the lymphoid cells present in gingiva from periodontitis patients, an in depth study involving the role of the immunoglobulin positive lymphocytes was investigated.^ Inflamed gingiva from four clinically defined periodontal disease states (mild gingivitis, severe gingivitis, periodontitis and severe periodontitis) were placed in gingival organ cultures. Class specific immunoglobulins were quantitated in gingival organ culture supernatants using an indirect sandwich technique. A significant difference in mean levels of IgA and IgG was seen between mild gingivitis and periodontitis (P < .00l, P = .001), as well as in IgG levels between periodontitis and severe periodontitis (P = .001). The predominance of IgG in gingival organ culture supernatants and the statistically significant findings that the overall mean levels of IgG between mild gingivitis and periodontitis (P = .014) and between severe periodontitis and periodontitis (P = .001) suggested a possible indicator of periodontal disease. The presence of IgG in gingival organ culture supernatants was shown to be a product of actively secreting plasma cells. The incorporation of radiolabelled amino acids into IgG was noted over a seven-day period with a peak response at day 4-5. The inhibition of IgG synthesis by cyclohexamide confirmed the contention that IgG was a product of de novo synthesis and not serum derived.^ The specificity of immunoglobulins derived from gingival organ cultures were studied using a whole bacterial agglutination test. Oral bacteria frequently cultured from periodontal patients were assessed for their ability to be agglutinated by gingival organ culture supernatants. A positive correlation of antibody titer and severity of disease was seen with five strains of Actinomyces viscosus, two of Actinomyces naeslundii and one Actinomyces israelii. The agglutination of bacteria was shown to be due to the specific interaction of immunoglobulin and cell-wall antigen. ^

A PHARMACOLOGICAL EVALUATION OF THE MECHANISM OF CYTOTOXICITY OF 9-BETA-D- XYLOFURANOSYLADENINE IN CHINESE HAMSTER OVARY CELLS

The biochemical determinants of cytotoxicity of the purine nucleoside analog, 9-(beta)-D-xylofuranosyladenine (xyl-A) were studied in wild-type Chinese hamster ovary cells and in nucleoside kinase deficient mutants. It was found that {('3)H}xyl-A was readily phosphorylated to the triphosphate level in both the wild-type and deoxycytidine kinase deficient mutant, but not by the adenosine kinase deficient cells. Values for the apparent Km and Vmax of this uptake process were 43.9 (mu)M and 118.7 nmol/min/10('9) cells, respectively. Cloning procedures indicated that the viability of CHO cells was decreased 90 per cent by a 5-hr incubation with 10 (mu)M xyl-A. However, the toxicity of xyl-A was increased 100-fold by the addition of a nontoxic concentration (10 (mu)M) of the adenosine deaminase inhibitor erythro-9-(2-hydroxy-3-nonyl)adenine (EHNA) to the medium. High-pressure liquid chromatographic analysis indicated that after 5 hr, the concentration of 9-(beta)-D-xylofuranosyladenine 5'-triphosphate (xyl-ATP) in cells incubated with xyl-A plus EHNA was 2.0 mM, four times greater than in those cells incubated with xyl-A alone. Incubation with xyl-A plus EHNA had no significant effect on the cellular concentrations of 5-phosphoribosyl-1-pyrophosphate after 1 hr whereas, treatment with 3'-dexoyadenosine (cordycepin) decreased the concentration of this metabolite. Determinations of the cellular nucleoside triphosphates indicated that under conditions that resulted in an intracellular accumulation of 500 (mu)M xyl-ATP, the endogenous concentrations of neither the ribonucleoside triphosphates nor deoxyribonucleoside triphosphates were significantly different from those of control cells. The ID(,50) for {('3)H}thymidine incorporation into DNA, 105 (mu)M xyl-ATP, was four-fold less than the ID(,50) for {('3)H}uridine incorporation into RNA suggesting that the process of DNA synthesis is more sensitive to the presence of xyl-ATP. When removed from exogenous xyl-A, CHO cells failed to recover their ability to synthesize RNA and DNA, although the intracellular xyl-ATP concentration decreased to less than 35 (mu)M. The selective inhibition of RNA synthesis by 6-azauridine did not prevent the expression of toxicity by xyl-ATP. However, the selective inhibition of DNA synthesis by ara-C significantly spared toxicity in cells that had accumulated an otherwise lethal concentration of xyl-ATP. It is shown that in cells which had accumulated 1.27 mM {('3)H}xyl-ATP, {('3)H}xyl-A was found to terminate cellular RNA chains at a frequency of 1.42 (mu)mol of {('3)H}xyl-A 3' termini per mol of mononucleotide. These results indicate that a general mechanism for the toxicity of xyl-A to CHO cells includes the cellular accumulation of xyl-ATP, which serves as a substrate for RNA synthesizing enzymes and subsequently is incorporated into nascent RNA transcripts as a chain terminator. A specific mechanism involving the premature termination of RNA primers required for the initiation of DNA synthesis is proposed to account for the inhibitory action of xyl-ATP on DNA synthesis. ^

UPTAKE, METABOLISM, AND METABOLIC EFFECTS OF THE ANTITUMOR TRICYCLIC NUCLEOSIDE, 3-AMINO-1, 5-DIHYDRO-5-METHYL-1-BETA-D-RIBOFURANOSYL-1, 4, 5, 6, 8 PENTAAZAACENAPHTHYLENE

The uptake, metabolism, and metabolic effects of the antitumor tricyclic nucleoside (TCN, NSC-154020) were studied in vitro. Uptake of TCN by human erythrocytes was concentrative, resulting mainly from the rapid intracellular phosphorylation of TCN. At high TCN doses, however, unchanged TCN was also concentrated within the erythrocytes. The initial linear rate of TCN uptake was saturable and obeyed Michaelis-Menten kinetics. TCN was metabolized chiefly to its 5'-monophosphate not only by human erythrocytes but also by wild-type Chinese hamster ovary (CHO) cells. In addition, three other metabolites were detected by means of high-performance liquid chromatography. The structures of these metabolites were elucidated by ultraviolet spectroscopy, infrared spectroscopy, mass spectrometry, and further confirmed by incubations with catabolic enzymes and intact wild-type or variant CHO cells. All were novel types of oxidative degradation products of TCN. Two are proposed to be (alpha) and (beta) anomers of a D-ribofuranosyl nucleoside with a pyrimido{4,5-c}pyridazine-4-one base structure. The third metabolite is most likely the 5'-monophosphate of the (beta) anomer. A CHO cell line deficient in adenosine kinase activity failed to phosphorylate either TCN or the (beta) anomer. No further phosphorylation of the 5'-monophosphates by normal cells occurred. Although the pathways leading to the formation of these TCN metabolites have not been proven, a mechanism is proposed to account for the above observations. The same adenosine kinase-deficient CHO cells were resistant to 500 (mu)M TCN, while wild-type cells could not clone in the presence of 20 (mu)M TCN. Simultaneous addition of purines, pyrimidines, and purine precursors failed to reverse this toxicity. TCN-treatment strongly inhibited formate or glycine incorporation into ATP and GTP of wild-type CHO cells. Hypoxanthine incorporation inhibited to a lesser degree, with the inhibition of incorporation into GTP being more pronounced. Although precursor incorporation into GTP was inhibited, GTP concentrations were elevated rather than reduced after 4-hr incubations with 20 (mu)M or 50 (mu)M TCN. These results suggested an impairment of GTP utilization. TCN (50 (mu)M) inhibited leucine and thymidine incorporation into HClO(,4)-insoluble material to 30-35% of control throughout 5-hr incubations. Incorporation of five other amino acids was inhibited to the same extent as leucine. Pulse-labeling assays (45 min) with uridine, leucine, and thymidine failed to reveal selective inhibition of DNA or protein synthesis by 0.05-50 (mu)M TCN; however, the patterns of inhibition were similar to those of known protein synthesis inhibitors. TCN 5'-monophosphate inhibited leucine incorporation by rabbit reticulocyte lysates; the inhibition was 2000 times less potent than that of cycloheximide. The 5'-monophosphate failed to inhibit a crude nuclear DNA-synthesizing system. Although TCN 5'-monophosphate apparently inhibits purine synthesis de novo, its cytotoxicity is not reversed by exogenous purines. Consequently, another mechanism such as direct inhibition of protein synthesis is probably a primary mechanism of toxicity. ^

Study of the cellular mechanism of Sunitinib mediated inactivation of activated hepatic stellate cells and its implications in angiogenesis

The development of hepatocellular carcinomas from malignant hepatocytes is frequently associated with intra- and peritumoral accumulation of connective tissue arising from activated hepatic stellate cells (HSC). Inhibition of receptor tyrosine kinase (RTK) signaling showed promise in the treatment of hepatocellular carcinoma. However, there is a lack of knowledge about the effects of RTK inhibitors on the tumor supportive cells. We performed in vitro experiments to study whether Sunitinib, a platelet-derived growth factor (PDGF) and vascular endothelial growth factor (VEGF) RTKs' inhibitor, could block both activated HSC functions and angiogenesis and thus prevent the progression of cirrhotic liver to hepatocellular carcinoma. In immortalized human activated HSC LX-2, treatment with Sunitinib 100 nM blocked collagen synthesis by 47%, as assessed by Sirius Red staining, attenuated HSC contraction by 65%, and reduced cell migration by 28% as evaluated using a Boyden's chamber, without affecting cell viability, measured by Trypan blue staining, and apoptosis, measured by propidium iodide (PI) incorporation assay. Our data revealed that Sunitinib treatment blocked the transdifferentiation of primary human HSC (hHSC) to activated myofibroblast-like cells by 65% without affecting hHSC apoptosis and migration. In in vitro angiogenic assays, Sunitinib 100 nM reduced endothelial cells (EC) ring formation by 46% and tube formation by 68%, and decreased vascular sprouting in aorta ring assay and angiogenesis in vascular bed of chick embryo. In conclusion, the present study demonstrates that the RTK inhibitor Sunitinib blocks the activation of HSC and angiogenesis suggesting its potential as a drug candidate in pathological conditions like liver fibrosis and hepatocellular carcinoma.

The ribosome as novel target for small regulatory RNAs

Small non-protein-coding RNA (ncRNA) molecules have been recognized recently as major contributors to regulatory networks in controlling gene expression in a highly efficient manner. While the list of validated ncRNAs that regulate crucial cellular processes grows steadily, not a single ncRNA has been identified that directly interacts and regulates the ribosome during protein biosynthesis (with the notable exceptions of 7SL RNA and tmRNA). All of the recently discovered regulatory ncRNAs that act on translation (e.g. microRNAs, siRNAs or antisense RNAs) target the mRNA rather than the ribosome. This is unexpected, given the central position the ribosome plays during gene expression. Furthermore it is strongly assumed that the primordial translation system in the ‘RNA world’ most likely received direct regulatory input from ncRNA-like cofactors. The fundamental question that we would like to ask is: Does the ‘RNA world still communicate’ with the ribosome? To address this question, we have analyzed the small ncRNA interactomes of ribosomes of prokaryotic (H. volcanii, S. aureus) and unicellular eukaryotic model organisms. Deep-sequencing and subsequent bioinformatic analyses revealed thousands of putative ribosome-associated ncRNAs. For a subset of these ncRNA candidates we have gathered experimental evidence that they are expressed in a stress-dependent manner and indeed directly target the ribosome. In the archaeon H. volcanii a tRNA-derived fragment was identified to target the small ribosomal subunit upon alkaline stress in vitro and in vivo. As a consequence of ribosome binding, this tRNA-fragment reduces protein synthesis by interfering with the peptidyl transferase activity. Our data reveal the ribosome as a novel target for small regulatory ncRNAs in all domains of life. Ribosome-bound ncRNAs are capable of fine tuning translation and might represent a so far largely unexplored class of regulatory sRNAs.