Bestimmung der Fluoreszenzkinetik von 5-Aminolävulinsäure-induziertem Protoporphyrin-IX an dreidimensionalen Organkulturen menschlicher Bronchialschleimhaut

Zielsetzung dieser Arbeit war es, die Fluoreszenzkinetik von 5-Aminolävulinsäure -induziertem Protoporphyrin-IX (PPIX) an dreidimensionalen Zellkulturen von humanem Bronchialepithel zu untersuchen, um Kenntnisse über den zeitlichen Verlauf der PPIX-Fluoreszenz in Normalgewebe nach unterschiedlich langer Expositionszeit mit 5- Aminolävulinsäure (5-ALA) zu erlangen. Da die Prognose von Patienten mit Bronchialkarzinomen in erster Linie vom Tumorstadium bei Diagnosestellung abhängig ist, ist es notwendig die Frühdiagnostik zu verbessern. Denkbar ist eine inhalative Anwendung von 5-ALA mit nachfolgender diagnostischer Bronchoskopie. Voraussetzung für diese Anwendung am Patienten und für weiterführende Untersuchungen sind Kenntnisse über die erforderliche 5-ALA-Kontaktzeit und Kenntnisse über den zeitlichen Verlauf der 5-ALA-induzierten PPIX- Fluoreszenz von Normalgewebe. Die Ergebnisse lassen sich wie folgt zusammenfassen: Ein 5-minütiger Kontakt mit 5-ALA ist ausreichend, um in normalem humanen Bronchialepithel detektierbare Mengen an PPIX-Fluoreszenz zu induzieren. Die Bildung von 5-ALA-induziertem PPIX erfolgt in Abhängigkeit der 5-ALA-Kontaktzeit. Die Höhe der Maximalintensität von 5-ALA-induzierter PPIX-Fluoreszenz ist ebenfalls von der 5-ALA-Kontaktzeit abhängig. Die Ergebnisse lassen folgende Schlussfolgerungen zu: Das Modell der dreidimensionalen Organkultur des humanen Bronchialepithels ist geeignet, um die 5-ALA-induzierte PPIX-Fluoreszenz zu untersuchen. Es konnte gezeigt werden, dass die 5-ALA-induzierte PPIX-Fluoreszenzkinetik in Kulturen des normalen humanen Bronchialepithels von der 5-ALA-Kontaktzeit abhängig ist. Die Resultate vorliegender Experimente sind am ehesten auf die Situation der topischen Anwendung (z.B. Inhalation) von 5-ALA übertragbar, da in diesem Modell kein intaktes Gefäßsystem zur Verfügung steht. Eine lokale Applikation für 5 oder 15 Minuten, die unter klinischen Bedingungen realisierbar wäre, ist nach den vorliegenden Ergebnissen für die PDD ausreichend

Kinetics of 5-aminolevulinic acid-induced fluorescence in organ cultures of bronchial epithelium and tumor

Background: 5-Aminolevulinic acid (5-ALA)-induced protoporphyrin IX (PPIX) fluorescence improves the differentiation of tumor and normal tissue in the bladder, skin and brain. Objective: The kinetics of 5-ALA-induced protoporphyrin IX (PPIX) fluorescence in organ cultures of normal human bronchial epithelium and cocultures of bronchial epithelium and tumor have been studied. Methods: Cultured biopsies of bronchial epithelium were exposed for 5 or 15 min, or continuously to 5-ALA. PPIX fluorescence was quantified for up to 300 min by spectroscopy. Cocultures of normal bronchial epithelium and a non-small-cell lung cancer cell line (EPLC-32M1) were incubated with 5-ALA. Space-resolved fluorescence microscopy was used to quantify PPIX fluorescence kinetics in the tumor and normal epithelium. Results: In cultures of normal epithelium, PPIX fluorescence kinetics were shown to depend on the duration of exposure to 5-ALA. There was a trend to higher fluorescence intensities with longer exposure times. In cocultures of bronchial epithelium and tumor, increases of fluorescence intensity were significantly greater in the tumor. Best tumor/normal tissue fluorescence ratios were found between 110 and 160 min after exposure to 5-ALA. Conclusion: Data obtained in this coculture system of bronchial epithelium and tumor is valuable to optimize modalities of fluorescence bronchoscopy for the diagnosis of early bronchial carcinoma. Copyright (C) 2002 S. Karger AG, Basel

Protein Kinase D regulates several aspects of development in Drosophila melanogaster

<p>Abstract</p> <p>Background</p> <p>Protein Kinase D (PKD) is an effector of diacylglycerol-regulated signaling pathways. Three isoforms are known in mammals that have been linked to diverse cellular functions including regulation of cell proliferation, differentiation, motility and secretory transport from the trans-Golgi network to the plasma membrane. In <it>Drosophila</it>, there is a single PKD orthologue, whose broad expression implicates a more general role in development.</p> <p>Results</p> <p>We have employed tissue specific overexpression of various PKD variants as well as tissue specific RNAi, in order to investigate the function of the PKD gene in <it>Drosophila</it>. Apart from a wild type (WT), a kinase dead (kd) and constitutively active (SE) <it>Drosophila </it>PKD variant, we also analyzed two human isoforms hPKD2 and hPKD3 for their capacity to substitute PKD activity in the fly. Overexpression of either WT or kd-PKD variants affected primarily wing vein development. However, overexpression of SE-PKD and PKD RNAi was deleterious. We observed tissue loss, wing defects and degeneration of the retina. The latter phenotype conforms to a role of PKD in the regulation of cytoskeletal dynamics. Strongest phenotypes were larval to pupal lethality. RNAi induced phenotypes could be rescued by a concurrent overexpression of <it>Drosophila </it>wild type PKD or either human isoform hPKD2 and hPKD3.</p> <p>Conclusion</p> <p>Our data confirm the hypothesis that <it>Drosophila </it>PKD is a multifunctional kinase involved in diverse processes such as regulation of the cytoskeleton, cell proliferation and death as well as differentiation of various fly tissues.</p

Deformation-induced phase transformation in a Co-Cr-W-Mo alloy studied by high-energy X-ray diffraction during in-situ compression tests

ickel-free Co-Cr-W-Mo alloys exhibit a very low or even negative stacking fault energy, and therefore a pronounced tendency towards a deformation-induced phase transformation of the metastable face-centered cubic (fcc) γ-phase to the hexagonal close-packed (hcp) low-temperature ε-phase. In order to analyze the phase transformation in-situ and to correlate it to an external strain, compression tests between 30 °C and 400 °C were performed in a deformation dilatometer simultaneously to high-energy X-ray diffraction. Hence, the elastic strains of the fcc unit cell during compression, the external loads for the onset of the phase transformation and the temperature-dependency could be determined. In the parent fcc γ-phase, the evolution of an $$ fiber texture as well as texture inheritance effects and a distinct variant selection could be observed. Further, for the investigated alloy composition it is demonstrated that the continuum concepts of i) a structural stretch tensor and ii) an invariant plane strain perfectly agree with the widely-accepted nucleation theory of ε-martensite formation in Co-Cr alloys via Shockley partial dislocations on every second {$111$}$_\gamma$ plane. Both, the observed transformation texture as well as crystallographic transformation strains reveal the importance of shear stresses in this system

Flies were raised in twelve-hour day-night cycle (left column; labelled with black and white bar) or in complete darkness (right column; black bar) and aged for several days at 25°C

<p><b>Copyright information:</b></p><p>Taken from "Protein Kinase D regulates several aspects of development in "</p><p>http://www.biomedcentral.com/1471-213X/7/74</p><p>BMC Developmental Biology 2007;7():74-74.</p><p>Published online 25 Jun 2007</p><p>PMCID:PMC1933421.</p><p></p> Time points shown: 3d, 0–3 days after hatching; 14d, 10–14 days after hatching. Enlargements of each 4 × 4 ommatida are shown to highlight the defects. A) Eyes of control flies: Note the regular architecture of ommatidia, which is independent of light and age. B) PKD-SE was overexpressed behind the morphogenetic furrow. In the light (left), rhabdomeres are elongated and sometimes fragmented or completely absent. In the dark (right) these phenotypes are less extreme but still detectable. They are largely independent of age. C) dsPKD was likewise induced in a light-dark cycle (left) and in the dark (right). Note degeneration of rhabdomeres in the light reared, 14 days old flies. The degeneration is less severe in younger flies or those kept in the dark. Genotypes are: A) gmr-Gal4/+; B) gmr-Gal4/PKD-SE; C) gmr-Gal4/+, UAS-dsPKD/+. Scale bar, 10 μm

A) Eyes of control flies have externally a smooth and regular appearance (left column)

<p><b>Copyright information:</b></p><p>Taken from "Protein Kinase D regulates several aspects of development in "</p><p>http://www.biomedcentral.com/1471-213X/7/74</p><p>BMC Developmental Biology 2007;7():74-74.</p><p>Published online 25 Jun 2007</p><p>PMCID:PMC1933421.</p><p></p> Tangential sections reveal the underlying, semi-crystalline architecture of the facets (center column). Seven photoreceptor cells can be distinguished by the centrally located rhabdomeres (arrow) that contain the light sensitive rhodopsin. Each facet is insulated by surrounding pigment cells that contain pigment granules (arrowhead). See enlargement in the right column. B) Overexpression of one or several copies PKD-SEcaused single black dots that have the size of a single facet (small arrow). Tangential sections reveal degeneration of the retina. The borders between ommatidia become blurred by many holes; also, fusion was observed. The pigment granules seem to clump and no longer encircle the rhabdomeres (arrowhead). The rhabdomeres are swollen and elongated; some are fragmented (arrow). Some rhabdomeres are absent and others degenerated. C) The defects remain largely unaltered when DIAP1 is overexpressed simultaneously, apart from a good rescue of the holes. D) Overexpression of dsPKD causes large black patches with a glossy surface (small arrow). Sections reveal massive amounts of cocci within the black patch (black arrowhead) underlying thinner lens (open arrow). Rhabdomeres are collapsed and degenerated (arrow); sporadic pigment granules can be seen (arrowhead). E) Concurrent overexpression of DIAP1 has little influence on the superficial appearance (arrow). However, the retina is somewhat more regular, which is reflected by the pigment granules that partly encircle the ommatidia (arrowhead). Moreover, rhabdomeres are well separated but many of them are degenerated (arrow). Genotypes are: A) wild type; B) gmr-Gal4/PKD-SE; C) UAS-DIAP1/+, gmr-Gal4/PKD-SE; D) gmr-Gal4/+, UAS-dsPKD/+; D) UAS-DIAP1/+, gmr-Gal4/+, UAS-dsPKD/+. Scale bar, 20 μm