Analysis of adenovirus trans-complementation-mediated gene expression controlled by melanoma-specific TETP promoter in vitro

BACKGROUND: Human adenoviruses (Ads) have substantial potential for clinical applications in cancer patients. Conditionally replicating adenoviruses (CRAds) include oncolytic adenoviruses in which expression of the immediate early viral transactivator protein E1A is controlled by a cancer cell-selective promoter. To enhance efficacy, CRAds are further armed to contain therapeutic genes. Due to size constraints of the capsid geometry, the capacity for packaging transgenes into Ads is, however, limited. To overcome this limitation, the employment of E1A-deleted replication-deficient viruses carrying therapeutic genes in combination with replication-competent CRAd vectors expressing E1A in trans has been proposed. Most trans-complementing studies involved transgene expressions from strong ubiquitous promoters, and thereby relied entirely on the cancer cell specificity of the CRAd vector. RESULTS: Here we tested the trans-complementation of a CRAd and a replication-deficient transgene vector containing the same cancer cell-selective promoter. Hereto, we generated two new vectors expressing IL-2 and CD40L from a bicistronic expression cassette under the control of the melanoma/melanocyte-specific tyrosinase enhancer tyrosinase promoter (TETP), which we previously described for the melanoma-specific CRAd vector AdDeltaEP-TETP. These vectors gave rise to tightly controlled melanoma-specific transgene expression levels, which were only 5 to 40-fold lower than those from vectors controlled by the nonselective CMV promoter. Reporter analyses using Ad-CMV-eGFP in combination with AdDeltaEP-TETP revealed a high level of trans-complementation in melanoma cells (up to about 30-fold), but not in non-melanoma cells, unlike the AdCMV-eGFP/wtAd5 binary vector system, which was equally efficient in melanoma and non-melanoma cells. Similar findings were obtained when replacing the transgene vector AdCMV-eGFP with AdCMV-IL-2 or AdCMV-CD40L. However, the combination of the novel AdTETP-CD40L/IL-2 vector with AdDeltaEP-TETP or wtAd5 gave reproducible moderate 3-fold enhancements of IL-2 by trans-complementation only. CONCLUSIONS: The cancer cell-selective TETP tested here did not give the expected enforceable transgene expression typically achieved in the Ad trans-complementing system. Reasons for this could include virus-mediated down regulation of limiting transcription factors, and/or competition for such factors by different promoters. Whether this finding is unique to the particular promoter system tested here, or also occurs with other promoters warrants further investigations

Macropinocytotic uptake and infection of human epithelial cells with species B2 adenovirus type 35

The human adenovirus serotype 35 (HAdV-35, short Ad35) causes kidney and urinary tract infections, and infects respiratory organs of immunocompromised individuals. Unlike other adenoviruses, Ad35 has a low seroprevalence which makes Ad35-based vectors promising candidates for gene therapy. Ad35 utilizes CD46 and integrins as receptors for infection of epithelial and hematopoietic cells. Here, we show that infectious entry of Ad35 into HeLa, human kidney HK-2 cells and normal human lung fibroblasts strongly depended on CD46 and integrins but not heparan sulfate, and variably required the large GTPase dynamin. Ad35 infections were independent of expression of the carboxy-terminal domain of AP180 which effectively blocks clathrin-mediated uptake. Ad35 infections were inhibited by small chemicals against the serine/threonine kinase Pak1 (p21-activated kinase), protein kinase C (PKC), sodium-proton exchangers, actin and acidic organelles. Remarkably, the F-actin inhibitor jasplakinolide, the Pak1 inhibitor IPA-3 or the sodium-proton exchange inhibitor EIPA blocked the endocytic uptake of Ad35. Dominant-negative proteins or small interfering RNAs against factors driving macropinocytosis, including the small GTPase Rac1, Pak1 or the Pak1 effector C-terminal binding protein 1 (CtBP1) potently inhibited Ad35 infection. Confocal laser scanning microscopy, electron microscopy and live cell imaging showed that Ad35 colocalized with fluid phase markers in large endocytic structures that were positive for CD46, alpha v integrins and also CtBP1. Our results extend earlier observations with HAdV-3 (Ad3), and establish macropinocytosis as an infectious pathway for species B human adenoviruses in epithelial and hematopoietic cells

Adenovirus triggers macropinocytosis and endosomal leakage together with its clathrin-mediated uptake

Adenovirus type 2 (Ad2) binds the coxsackie B virus Ad receptor and is endocytosed upon activation of the αv integrin coreceptors. Here, we demonstrate that expression of dominant negative clathrin hub, eps15, or K44A-dynamin (dyn) inhibited Ad2 uptake into epithelial cells, indicating clathrin-dependent viral endocytosis. Surprisingly, Ad strongly stimulated the endocytic uptake of fluid phase tracers, coincident with virus internalization but without affecting receptor-mediated transferrin uptake. A large amount of the stimulated endocytic activity was macropinocytosis. Macropinocytosis depended on αv integrins, PKC, F-actin, and the amiloride-sensitive Na+/H+ exchanger, which are all required for Ad escape from endosomes and infection. Macropinocytosis stimulation was not a consequence of viral escape, since it occurred in K44A-dyn–expressing cells. Surprisingly, 30–50% of the endosomal contents were released into the cytosol of control and also K44A-dyn–expressing cells, and the number of fluid phase–positive endosomes dropped below the levels of noninfected cells, indicating macropinosomal lysis. The release of macropinosomal contents was Ad dose dependent, but the presence of Ad particles on macropinosomal membranes was not sufficient for contents release. We conclude that Ad signaling from the cell surface controls the induction of macropinosome formation and leakage, and this correlates with viral exit to the cytosol and infection

Cell Cycle-Dependent Kinase Cdk9 Is a Postexposure Drug Target against Human Adenoviruses

Human adenoviruses (HAdVs) infect respiratory, gastrointestinal, and urinary tracts and give rise to eye infections and epidemic keratoconjunctivitis (EKC). They persist in lymphoid tissue and cause morbidity and mortality in immunocompromised people. Treatments with significant postexposure efficacy are not available. Here, we report that inhibition of the cell cycle-dependent kinase 9 (Cdk9) by RNA interference, or the compound flavopiridol, blocked infections with HAdV-C2/5, EKC-causing HAdV-D8/37, and progeny formation in human corneal epithelial and cancer cells. Flavopiridol abrogated the production of the immediate early viral transactivating protein E1A without affecting nuclear import of viral DNA. In morphometric plaque assays, the compound exhibited antiviral efficacy in both pre- and postexposure regimens with therapeutic indexes exceeding 10. The study identifies Cdk9 as a postexposure drug target against adenovirus infections in vitro and suggests that the clinically tested anticancer drug flavopiridol is a candidate for treating adenoviral EKC or adenovirus emergence upon immune suppression

Validation of the High Performance Conduction-Cooled Prototype LTS Pulse Coil for UPS-SMES

A conduction-cooled low temperature superconducting (LTS) pulse coil has been developed as a key technology for UPS-SMES. We have been developing a 1 MW, 1 s UPS-SMES for a protection from a momentary voltage drop and an instant power failure. A conduction-cooled LTS pulse coil has excellent characteristics, which are adequate for a short-time uninterruptible power supply (UPS). The LTS coil has better cost performance over the HTS coil at present and the conduction cooling has higher reliability and easier operation than the conventional cooling schemes such as pool boiling with liquid helium or forced flow of supercritical helium. To demonstrate the high performances of the LTS pulse coil, we have fabricated a prototype coil with stored energy of 100 kJ and have conducted cooling and excitation tests. The successful performance test results including current shut-off test with a time constant of 1.3 s and repeated excitation of a triangular waveform with high ramp rate are reporte

Measurement of tau polarization in e+ e- annihilation at sqrt{s}=58 GeV

The polarization of tau leptons in the reaction e+ e- --> tau+ tau- has been measured using a e+e- collider, TRISTAN, at the center-of-mass energy of 58 GeV. From the kinematical distributions of daughter particles in tau --> e nu nu-bar, mu nu nu-bar, rho nu or pi(K) nu decays, the average polarization of tau- and its forward-backward asymmetry have been evaluated to be 0.012 +- 0.058 and 0.029 +- 0.057, respectively.Comment: 18 pages, 5 figure

Weltrohstoffversorgung: Konflikt oder Kooperation?

Unter dem Eindruck der jüngsten Preishausse auf den internationalen Rohstoffmärkten wird nunmehr in vielen Ländern befürchtet, daß die Welt an der Schwelle zunehmender Schwierigkeiten bei der Versorgung mit mineralischen und landwirtschaftlichen Rohstoffen einschließlich Nahrungsmitteln steht. Der kräftige Preisanstieg bei diesen Rohstoffen war jedoch überwiegend konjunkturell bedingt. Bei Getreide verursachten Mißernten unerwartete Produktionsausfälle größeren Ausmaßes. Wirksame Preisabsprachen zwischen Produzentenländern hat es, anders als bei Erdöl, entgegen einer verbreiteten Meinung nicht gegeben. Längerfristig wird sich das Preisniveau auf den Rohstoffmärkten, wenn auch nicht drastisch, so doch kontinuierlich erhöhen. Preisfluktuationen um den Trend werden weiterhin diese Märkte kennzeichnen. Ernsthafte Rohstoffverknappungen brauchen für den Rest dieses Jahrhunderts nicht erwartet zu werden. So übersteigen z. B. im Falle zahlreicher nicht-regenerierbarer NE-Metalle die bekannten Weltreserven deutlich den Weltbedarf; bei tropischen Ernährungsgütern und bei Getreide ist der Spielraum für eine nachhaltige Erhöhung der Flächenerträge noch sehr groß, vor allem in Entwicklungsländern. Die Wahrscheinlichkeit für die Bildung von Produzentenkartellen in der Zukunft ist gering. Käme es dennoch zu Kartellabsprachen, so wären diese bestenfalls kurzfristig erfolgreich. Das gleiche gilt für Exportbeschränkungen, die in Zeiten knappen Angebots sensibler Produkte (z. B. Getreide) den internen Preisanstieg dämpfen sollen. Angesichts der verbreiteten Knappheitspsychose kann nicht ausgeschlossen werden, daß es im Falle von temporären Angebotsdefiziten zu Überreaktionen kommt, die erhebliche Gefahren für den freien und multilateralen Welthandel in sich bergen. Solchen Gefahren kann nur erfolgreich durch partnerschaftliche Zusammenarbeit, nicht jedoch durch nationale Alleingänge, begegnet werden. Erforderlich sind ein international vereinbartes System von Verhaltensregeln zur Sicherung des undiskriminierten Zugangs zu Rohstoffmärkten bei angemessenen Preisen, die koordinierte Bildung von Rohstoffvorräten, internationale Rohstoffabkommen zwischen Produzenten- und Verbraucherländern, Regelungen zur Verbesserung des allgemeinen Investitionsklimas im Rohstoffbereich und der Abbau der Handelsschranken der Industrieländer gegenüber Exporten landwirtschaftlicher Produkte sowie rohstoff- und/oder arbeitsintensiver Halb- und Fertigwaren aus Entwicklungsländern. --

Welthandel und Strukturanpassung in den Industrieländern

Ziel der sechsten GATT-Runde ist es, die Schranken im Welthandel weiter abzubauen. Anders als bei den fünf früheren GATT-Runden herrscht dieses Mal in vielen Ländern Skepsis gegenüber einer weiteren Liberalisierung, weil man die Risiken, die mit zunehmendem Importwettbewerb verbunden sein können, höher bewertet als die möglichen Vorteile der Liberalisierung. Das Aufrechterhalten von Handelsschranken kostet Ressourcen. Die Protektion wenig effizienter Industrien in den wohlhabenden Ländern hält deren Einkommen unter dem Niveau, das sie sonst erreichen könnten und verzögert das Tempo der Industrialisierung in den Entwicklungsländern. Die ökonomischen Argumente zugunsten eines freieren Handels sind stark und weitgehend unanfechtbar. Handelsbeschränkungen sind zudem politisch belastend. Es gibt eine Reihe staatlicher Maßnahmen, die geeignet sind, die Schocks einer Anpassung an wirtschaftliche Veränderungen zu mindern. Interne Anpassungmaßnahmen spielen daher eine bedeutende Rolle in der künftigen Handelspolitik. Sehr zweifelhaft ist, ob alle Möglichkeiten für wirksame Anpassungsmaßnahmen bereits ausgeschöpft sind. In der Textilindustrie, der Stahlindustrie und im Kohlenbergbau existieren nach allgemeiner Meinung hartnäckige Anpassungsprobleme, die besondere und permanente Schutzmaßnahmen gegen den Importwettbewerb verlangen. Obwohl es viele Anstrengungen gegeben hat, den Status quo aufrechtzuerhalten, hat in allen drei Fällen die Dynamik des Strukturwandels die Lage grundlegend verändert. Diese Entwicklungstendenzen erlauben den Schluß, daß weitere Schritte zur Liberalisierung auch in den schwierigsten Fällen nicht nur wünschenswert wären, sondern wahrscheinlich durch den immanenten Strukturwandel in der Wirtschaft erleichtert werden. Eine Veränderung ist selten bequem, selbst dann nicht, wenn sie zu Verbesserungen führt. Für die Politik darf die relevante Frage nicht sein, wie man eine Anpassung an veränderte Umstände verlangsamen oder verhindern könnte. Sie lautet vielmehr, wie man die Chancen, die ein freierer Handel bietet, dadurch nutzen kann, daß man die Anpassung erleichtert und ihre Kosten minimiert. --

Cone pigments in a North American marsupial, the opossum (Didelphis virginiana)

Only two of the four cone opsin gene families found in vertebrates are represented in contemporary eutherian and marsupial species. Recent genetic studies of two species of South American marsupial detected the presence of representatives from two of the classes of cone opsin genes and the structures of these genes predicted cone pigments with respective peaks in the ultraviolet and long-wavelength portions of the spectrum. The Virginia opossum (Didelphis virginiana), a profoundly nocturnal animal, is the only marsupial species found in North America. The prospects for cone-based vision in this species were examined through recordings of the electroretinogram (ERG), a commonly examined retinal response to photic stimulation. Recorded under flickering-light conditions that elicit signals from cone photoreceptors, the spectral sensitivity of the opossum eye is well accounted for by contributions from the presence of a single cone pigment having peak absorption at 561–562 nm. A series of additional experiments that employed various chromatic adaptation paradigms were conducted in a search for possible contributions from a second (short-wavelength sensitive) cone pigment. We found no evidence that such a mechanism contributes to the ERG in this marsupial