Characterization of the Bafilomycin Biosynthetic Gene Cluster from Streptomyces lohii

Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/96305/1/301_ftp.pdfhttp://deepblue.lib.umich.edu/bitstream/2027.42/96305/2/cbic_201200743_sm_miscellaneous_information.pd

Tailoring of polar and nonpolar ZnO planes on MgO (001) substrates through molecular beam epitaxy

Polar and nonpolar ZnO thin films were deposited on MgO (001) substrates under different deposition parameters using oxygen plasma-assisted molecular beam epitaxy (MBE). The orientations of ZnO thin films were investigated by in situ reflection high-energy electron diffraction and ex situ X-ray diffraction (XRD). The film roughness measured by atomic force microscopy evolved as a function of substrate temperature and was correlated with the grain sizes determined by XRD. Synchrotron-based X-ray absorption spectroscopy (XAS) was performed to study the conduction band structures of the ZnO films. The fine structures of the XAS spectra, which were consistent with the results of density functional theory calculation, indicated that the polar and nonpolar ZnO films had different electronic structures. Our work suggests that it is possible to vary ZnO film structures from polar to nonpolar using the MBE growth technique and hence tailoring the electronic structures of the ZnO films

Regulation von Membrantransportvorgängen durch Lipide und Proteine

Title and directory Abstract Introduction Aims of the studies Materials and methods Results Discussion Conclusions Bibliography Appendix List of publicationsSpecific membrane lipids have been proposed to regulate protein sorting during the exo- and endocytic pathways at multiple stages. This includes the formation of membrane microdomains, and membrane shape changes induced by recruitment of peripheral proteins. Here we have addressed both mechanisms in two distinct experimental systems: (1) the recycling of synaptic vesicles (SVs) at nerve terminals and (2) membrane deformation at the Golgi/endosomal boundary. SVs in the central nervous system upon stimulation undergo rapid calcium-triggered exo-endocytic cycling within the nerve terminal which at least in part depends on components of the clathrin- and dynamin-dependent endocytosis machinery. How exocytic SV fusion and endocytic retrieval are temporally and spatially coordinated is still an open question. One possibility is that specialized membrane microdomains characterized by their high content in membrane cholesterol may assist in the spatial coordination of synaptic membrane protein recycling. Quantitative proteomic analysis of detergent-resistant lipid microdomains (DRMs) isolated from rat brain synapses or cholesterol-depleted control samples by liquid chromatography tandem mass spectrometry identified a total of 159 proteins. Among these 122 proteins were classified as cholesterol-dependent DRM or DRM-associated proteins, many of which with proven or hypothesized functions in exo-endocytic vesicle cycling including clathrin, the clathrin adaptor complex AP-2, and a variety of SV proteins. In agreement with this SV membrane and endocytic proteins display a partial resistance to extraction with cold Triton X-100 in cultured rat hippocampal neurons where they co-localize with labeled cholera toxin B, a marker for cholesterol-enriched DRMs. Moreover, SV proteins form cholesterol- dependent complexes in CHAPS-extracted synaptic membrane lysates. Membrane traffic at the Golgi/endosomal boundary requires the action of Arfs (ADP ribosylation factors), NH2-terminal myristoylated proteins that belong to the Ras superfamily of small GTP binding proteins. We demonstrate that bothwild type myristoylated and non-myristoylated recombinant Arf1 and Arf6 can bind to liposomes and bend them into tubular structures as observed by electron and video microscopy. Mutations of hydrophobic residues within the amphipathic helices abolish their liposome binding or tubulation capacity. Co- overexpression of Arf1-EGFP and FAPP-PH domain, a PI4P-binding protein module, in COS7 cells produces enormous tubules from a perinuclear compartment. In comparison, Arf1 NH2-terminal hydrophobic residue mutants show a cytosolic localization similar to GDP-locked Arf1 T31N. The NH2-terminal amphipathic helix itself is sufficient to drive membrane curvature generation when overexpressed in COS7 cells. These data identify a novel GTP-controlled switch that regulates membrane deformation during budding of coated vesicles and tubules at the Golgi/endosomal boundary.Möglicherweise können bestimmte Membranlipide die Proteinsortierung während der Exo- und Endocytose auf verschiedenen Ebenen regulieren. Dies könnte z.B. durch die Formierung sog. Membranmikrodomänen aber auch durch Veränderung in der Membrangestalt, induziert durch die Bindung peripherer Proteine, geschehen. In dieser Arbeit wurden beide Mechanismen durch zwei verschiedene experimentelle Systeme untersucht: 1) Recycling synaptischer Vesikel an Nervenendigung und 2) Membranveränderungen an der Golgi/Endosomen-Grenze. Synaptische Vesikel im zentralen Nervensystem werden nach Stimulierung einem schnellen, Calcium-induzierten, exo- und endocytischen Zyklus unterworfen. Dieser hängt zumindest teilweise von Komponenten der Clathrin und Dynamin- abhängigen Endocytose-Maschinerie ab. Wie die Vesikelfusion und die Aufnahme zeitlich und räumlich koordiniert werden ist ungewiss. Eine Möglichkeit ist, dass spezialisierte Membrandomänen, die durch einen hohen Gehalt an Cholesterin charakterisiert sind, die räumliche Voraussetzung schaffen, um synaptische Membranproteine aufzunehmen. Quantitative Proteinanalysen von Detergenz-resistenten Lipidmikrodomänen (DRMs) aus Rattengehirnsynapsen oder aus Cholesterin depletierten Kontrollen, ergaben nach massenspektroskopischen Untersuchungen 159 Proteine. Darunter waren 122 Proteine als Cholesterin- abhängige DRM oder DRM-assoziierte Proteine aufgeführt, viele davon mit bewiesener Funktion im exo-und endocytischen Vesikelkreislauf. Darunter waren Clathrin, der Clathrin Adaptor Komplex AP-2 und verschiedene synaptische Vesikelproteine. In Übereinstimmung damit sind Vesikelmembranproteine und endocytierte Proteine gegenüber einer Extraktion mit kaltem Triton in kultivierten Rattenhippocampus-Neuronen resistent. Hier kolokalisieren sie mit markiertem Choleratoxin B, einem Protein das spezifisch für cholesterinhaltige DRMs ist. Darüberhinaus formen Vesikelproteine Cholesterin abhängige Komplexe in CHAPS extrahierten Membranlysaten. Membranverkehr an der Golgi/endosomalen Grenze benötigt ARFs (ADP ribosylation factors), NH2- terminal myristoylierte Proteine, die zur Ras Superfamilie kleiner GTP-bindender Proteine gehören. Wir zeigen Normales und nicht myristoyliertes rekombinantes Arf1 und Arf6 können an Liposomen binden und sie zu tubulären Strukuren krümmen, wie man im Elektronen und Videomikroskop sehen konnte. Mutationen hydrophober Bestandteile der amphipathischen Helix zerstörten die Fähigkeit Liposomen zu krümmen. Überexpression von Arf1-EGFP und FAPP-PH, einem PI(4)P bindenden Proteinbaustein, in COS7 Zellen ergab enorme tubuläre Strukturen aus einem perinukleärem Kompartiment. Dagegen zeigten Arf1 Mutanten deren NH2 terminale hydrophobische Bestandteile verändert wurden, eine zytosolische Lokalisation, die ähnlich der GDP-gesperrten Arf1 Mutante T31N war. Die NH2 terminale amphipathische Helix selbst genügt, um bei Überexpression in Cos7 Zellen Membrankrümmung zu erreichen. Diese Daten identifizieren einen neuen GTP- kontrollierten Schalter, der die Membrandeformation während der Vesikelausknospung an der Golgi/endosomalen Grenze regulieren kann

Activated hepatic stellate cells promote hepatocellular carcinoma development in immunocompetent mice

National Key Sci-Tech Special Project of China [2008ZX10002-019]; National Natural Science Foundation of China [30872484]Activated hepatic stellate cells (HSCs) play a central role in the hepatic fibrosis and cirrhosis. Recently, HSCs were reported to have strong immune modulatory activities. However, the role of HSCs in hepatocellular carcinoma (HCC) remains unclear. In this study, we showed that HSCs could promote HCC growth both in vitro and in vivo. We examined the HSC-mediated inhibition of T-cell proliferation and the ability of conditioned medium from activated HSCs to promote the growth of murine HCC cell lines in vitro. We also assessed the immune suppression by HSCs during the development of HCC in immunocompetent mice. Cotransplantation of HSCs promoted HCC growth and progression by enhancing tumor angiogenesis and tumor cell proliferation and by creating an immunosuppressed microenvironment. Cotransplanted HSCs inhibited the lymphocyte infiltration in tumors and the spleens of mice bearing tumors, induced apoptosis of infiltrating mononuclear cells, and enhanced the expression of B7H1 and CD4(+)CD25(+) Treg cells. The immune modulation by HSCs seemed to be systemic. In conclusion, our data provide new information to support an integral role for HSCs in promoting HCC progression in part via their immune regulatory activities, and suggest that HSCs may serve as a therapeutic target in HCC