Untersuchungen zur Expression und Lokalisation der respiratorischen Proteine Neuroglobin (Ngb) und Cytoglobin (Cygb) in Säugern

Die vorliegende Dissertation beinhaltet Untersuchungen zur Expression und Funktion der respiratorischen Proteine Neuroglobin (Nbg) und Cytoglobin (Cygb) in Vertebraten. rnrnUm die Expression der Globine während der Entwicklung des Säugerhirns zu untersuchen, wurden die Hirne von Maus-Embryonen ab dem Fötalstadium MF10 bis zum Tag eins nach der Geburt (T1) mit Adulttieren verglichen. Quantifiziert wurde sowohl die mRNA- als auch die Protein-Expression. Beide Globine zeigten im Verlauf der Entwicklung einen stetigen Anstieg der mRNA-Expression, wobei Ngb zu Beginn in zehnfach höherer Konzentration vorlag und im zeitlichen Verlauf einen 130-fachen Anstieg zeigte. Cygb zeigte lediglich einen 16-fachen Anstieg bis zum Adultstadium. Auf Proteinebene konnte die Expressionszunahme beider Globine im Laufe der Entwicklung bestätigt werden. Weder in den hypoxieresistenten Frühembryonalstadien noch während der mit Sauerstoff-Stress verbundenen Geburt zeigte sich ein Expressionsmaximum. Dies spricht gegen eine Globin-Funktion in der Oxidanz-Abwehr. Eher ist zumindest Ngb mit der Reifung der Neurone und dem damit einhergehenden, gesteigerten oxidativen Stoffwechsel assoziiert.rnrnDes Weiteren sollte die zelluläre und intrazelluläre Lokalisation beider Globine anhand einer primären Zellkultur aus dem Hippocampus pränataler Ratten und in immortalen Zelllinien untersucht werden. Neuroglobin wurde dabei nur in Neuronen, nicht jedoch in Gliazellen nachgewiesen. Das Färbemuster war in allen Ngb-exprimierenden Zellen zytoplasmatisch. Cytoglobin wurde in der Primärkultur in den Neuronen jedoch ebenso in den mit anti-GFAP markierten Gliazellen beobachtet. In beiden Zellpopulationen war auch der Kern durch das CyGB-Antiserum markiert. rnEine genauere Untersuchung der intrazellulären Lokalisation sollte durch die Transfektion von Globin-pEGFP-Fusionsproteinen erfolgen. Nach Transfektion der Fusionskonstrukte wurde die GFP-Färbung bei beiden Globinen sowohl im Zytoplasma als auch im Kern beobachtet. Eine rein nukleäre Lokalisation, die insbesondere für Cygb von anderen Autoren postuliert wurde, konnte somit ausgeschlossen werden. rnrnIn primären Zellkulturen aus Cerebellum und Kortex, die mit Hilfe von Paraquat oxidativem Stress ausgesetzt wurden, wurde der Verlauf der Globin-mRNA-Expression mit dem unregulierten 18s rRNA-Referenzgen und mit den Antioxidanz-Enzymen Cu-Zn-SOD und Gpx verglichen. Neuroglobin zeigte einen Expressionsverlauf ähnlich dem der beiden Antioxidanz-Enzyme, jedoch liegt seine mRNA im Hirngewebe in hundertfach niedrigerer Menge als Cu-Zn-SOD und Gpx vor. Cytoglobin zeigte keine Veränderung der Expression. Eine Funktion der Globine im Sinne einer ROS-Abwehr kann aus den Befunden nicht abgeleitet werden. rnrnUntersuchungen von Tumor und Normalgewebe mittels eines cDNA-Cancer-Arrays zeigten, dass NGB in Tumoren verschiedenen Ursprungs nicht exprimiert wird, CyGB dagegen keine Änderung seiner Expression in Tumor versus Normalgewebe erfährt. Eine Induktion der beiden Globine z.B. durch Hypoxie in soliden Tumoren kann daher ausgeschlossen werden.rn

Identification and characterisation of Stx5 a novel interactor of VLDL-R affecting its intracellular trafficking and processing

We identified syntaxin 5 (Stx5), a protein involved in intracellular vesicle trafficking, as a novel interaction partner of the very low density lipoprotein (VLDL)-receptor (VLDL-R), a member of the LDL-receptor family. In addition, we investigated the effect of Stx5 on VLDL-R maturation, trafficking and processing. Here, we demonstrated mutual association of both proteins using several in vitro approaches. Furthermore, we detected a special maturation phenotype of VLDL-R resulting from Stx5 overexpression. We found that Stx5 prevented Golgi-maturation of VLDL-R, but did not cause accumulation of the immature protein in ER to Golgi compartments, the main expression sites of Stx5. Rather more, abundantly present Stx5 was capable of translocating ER-/N-glycosylated VLDL-R to the plasma membrane, and thus was insensitive to BFA treatment and incubation at low temperature. Based on our findings, we postulate that Stx5 can directly bind to the C-terminal domain of VLDL-R, thereby influencing the receptor’s glycosylation, trafficking and processing characteristics. Resulting from that, we further suggest that Stx5, which is highly expressed in neurons along with VLDL-R, might play a role in modulating the receptor’s physiology by participating in a novel/undetermined alternative pathway bypassing the Golgi apparatus.

Drug delivery, entry and intracellular trafficking of polymeric nanoparticles

Polymere Nanopartikel sind kleine Teilchen, die vielseitige Einsatzmöglichkeiten für den Transport von Wirkstoffen bieten. Da Nanomaterialien in diesen biomedizinischen Anwendungen oft mit biologischen Systemen in Berührung kommen, erfordert das eine genaue Untersuchung ihrer gegenseitigen Wechselwirkungen. In diesem speziellen Forschungsgebiet, welches sich auf die Interaktionen von Nanomaterialien mit biologischen Komponenten konzentriert, wurde bereits eine Vielzahl verschiedener Nanopartikel-Zell-Interaktionen (z. B. Nanotoxizität, Wirkstofftransport-mechanismen) analysiert. Bezüglich der Untersuchungen zu nanopartikulären Wirkstofftransport-mechanismen ist es im Allgemeinen akzeptiert, dass ein erfolgreicher zellulärer Transport hauptsächlich von der Aufnahme des Nanotransporters abhängt. Deshalb analysieren wir in dieser Arbeit (1) den Wirkstofftransportmechanismus für biologisch-abbaubare eisenhaltige Poly-L-Milchsäure Nanopartikel (PLLA-Fe-PMI) sowie (2) die Aufnahmemechanismen und die intrazellulären Transportwege von nicht-abbaubaren superparamagnetischen Polystyrolnanopartikeln (SPIOPSN). rnIn dieser Arbeit identifizieren wir einen bisher unbekannten und nicht-invasiven Wirkstoff-transportmechanismus. Dabei zeigt diese Studie, dass der subzelluläre Transport der nanopartikulärer Fracht nicht unbedingt von einer Aufnahme der Nanotransporter abhängt. Der identifizierte Arzneimitteltransportmechanismus basiert auf einem einfachen physikochemischen Kontakt des hydrophoben Poly-L-Milchsäure-Nanopartikels mit einer hydrophoben Oberfläche, wodurch die Freisetzung der nanopartikulären Fracht ausgelöst wird. In Zellexperimenten führt die membranvermittelte Freisetzung der nanopartikulären Fracht zu ihrem sofortigen Transport in TIP47+- und ADRP+- Lipidtröpfchen. Der Freisetzungsmechanismus („kiss-and-run") kann durch die kovalente Einbindung des Frachtmoleküls in das Polymer des Nanopartikels blockiert werden.rnWeiterhin wird in Langzeitversuchen gezeigt, dass die Aufnahme der untersuchten polymeren Nanopartikel von einem Makropinozytose-ähnlichen Mechanismus gesteuert wird. Im Laufe dieser Arbeit werden mehrere Faktoren identifiziert, die in diesem Aufnahmemechanismus eine Rolle spielen. Darunter fallen unter anderem die kleinen GTPasen Rac1 und ARF1, die die Aufnahme von SPIOPSN beeinflussen. Darauffolgend werden die intrazellulären Transportwege der Nanopartikel untersucht. Mit Hilfe eines neuartigen Massenspektrometrieansatzes wird der intrazelluläre Transport von nanopartikelhaltigen endozytotischen Vesikeln rekonstruiert. Intensive Untersuchungen identifizieren Marker von frühen Endosomen, späten Endosomen/ multivesikulären Körpern, Rab11+- Endosomen, Flotillin-Vesikeln, Lysosomen und COP-Vesikeln. Schließlich wird der Einfluss des lysosomalen Milieus auf die Proteinhülle der Nanopartikel untersucht. Hier wird gezeigt, dass die adsorbierte Proteinhülle auf den Nanopartikeln in die Zelle transportiert wird und anschließend im Lysosom abgebaut wird. rnInsgesamt verdeutlicht diese Arbeit, dass die klassische Strategie des nanopartikulären und invasiven Wirkstofftransportmechanismuses überdacht werden muss. Weiterhin lässt sich aus den Daten schlussfolgern, dass polymere Nanopartikel einem atypischen Makropinozytose-ähnlichen Aufnahmemechanismus unterliegen. Dies resultiert in einem intrazellulären Transport der Nanopartikel von Makropinosomen über multivesikuläre Körperchen zu Lysosomen.rn

Comparative functional analysis of factors controlling glial differentiation in Drosophila and mouse

The present study is a comparative functional analysis of three factors controlling glial differentiation in mouse (Fyn Src kinase, hnRNPF/H and NG2) and their homologues in Drosophila (Src42A and 64B, Glorund and Kon-tiki (Kon)). In Drosophila, mutations in any of these genes were not associated with major embryonic neurodevelopmental phenotypes. Src kinases and Glorund were shown to be ubiquitously expressed, whereas kon mRNA showed selective expression in muscles as well as in central and peripheral glia. Kon was also shown to be expressed in L3 larvae with high levels of protein accumulation at the neuromuscular junction (NMJ) and in muscles in the form of speckles. Knockdown of kon in glia resulted in NMJ phenotypes, mainly characterized by a significant increase in bouton number and a reduction in α-Konecto staining intensity at the NMJ. From the three glial layers ensheathing the peripheral nervous system, subperineurial glial showed to be the one contributing the most to kon knockdown dependent NMJ phenotypes, while perineurial glia only had a minor role. The knockdown of kon in glia also showed to affect Glutamate receptor subunit (α-GluRIIA) clustering in the postsynapse, same as microtubule arrangement in the presynapse, as seen by α-Futsch pattern interruptions and alterations. kon knockdown in glia also resulted in impaired axonal transport, as seen by the accumulation of Bruchpilot-positive vesicles along the nerves, abnormal formation of neuronal derived protrusions and swellings, filled with vacuole-like structures. Glia number along the peripheral nerves is also reduced as consequence of kon knockdown. Muscle derived Kon was shown to accumulate at the NMJ and play a role in bouton consolidation and to interfere with phagocytosis of ghost boutons. NMJ bouton and branch number was also significantly increased in Kon overexpression in glia. The overexpression of Kon in glia also resulted in a massive elongation of the ventral nerve cord, which served in a suppressor screen to identify intracellular interaction partners of Kon in glia. It was shown that Kon is processed in glia and preliminary results indicate that the metalloendopeptidase Kuzbanian (the fly homologue of ADAM10) may play a role in the shedding of Konecto. In the present work, Kon is shown as a multifunctional gene with various roles in glia-neuron and glia-neuron-muscle interaction.

A randomized, controlled clinical trial on the clinical, microbiological, and staining effects of a novel 0.05% chlorhexidine/herbal extract and a 0.1% chlorhexidine mouthrinse adjunct to periodontal surgery

BACKGROUND: Chlorhexidine (CHX) rinsing after periodontal surgery is common. We assessed the clinical and microbiological effects of two CHX concentrations following periodontal surgery. MATERIALS AND METHODS: In a randomized, controlled clinical trial, 45 subjects were assigned to 4 weeks rinsing with a 0.05 CHX/herbal extract combination (test) or a 0.1% CHX solution. Clinical and staining effects were studied. Subgingival bacteria were assessed using the DNA-DNA checkerboard. Statistics included parametric and non-parametric tests (p<0001 to declare significance at 80% power). RESULTS: At weeks 4 and 12, more staining was found in the control group (p<0.05 and p<0.001, respectively). A higher risk for staining was found in the control group (crude OR: 2.3:1, 95% CI: 1.3 to 4.4, p<0.01). The absolute staining reduction in the test group was 21.1% (9 5% CI: 9.4-32.8%). Probing pocket depth (PPD) decreases were significant (p<0.001) in both groups and similar (p=0.92). No rinse group differences in changes of bacterial counts for any species were found between baseline and week 12. CONCLUSIONS: The test CHX rinse resulted in less tooth staining. At the study endpoint, similar and high counts of periodontal pathogens were found.

Intraneuronal angiotensinergic system in rat and human dorsal root ganglia

To elucidate the local formation of angiotensin II (Ang II) in the neurons of sensory dorsal root ganglia (DRG), we studied the expression of angiotensinogen (Ang-N)-, renin-, angiotensin converting enzyme (ACE)- and cathepsin D-mRNA, and the presence of protein renin, Ang II, Substance P and calcitonin gene-related peptide (CGRP) in the rat and human thoracic DRG. Quantitative real time PCR (qRT-PCR) studies revealed that rat DRG expressed substantial amounts of Ang-N- and ACE mRNA, while renin mRNA as well as the protein renin were untraceable. Cathepsin D-mRNA and cathepsin D-protein were detected in the rat DRG indicating the possibility of existence of pathways alternative to renin for Ang I formation. Angiotensin peptides were successfully detected with high performance liquid chromatography and radioimmunoassay in human DRG extracts. In situ hybridization in rat DRG confirmed additionally expression of Ang-N mRNA in the cytoplasm of numerous neurons. Intracellular Ang II staining could be shown in number of neurons and their processes in both the rat and human DRG. Interestingly we observed neuronal processes with angiotensinergic synapses en passant, colocalized with synaptophysin, within the DRG. In the DRG, we also identified by qRT-PCR, expression of Ang II receptor AT(1A) and AT(2)-mRNA while AT(1B)-mRNA was not traceable. In some neurons Substance P and CGRP were found colocalized with Ang II. The intracellular localization and colocalization of Ang II with Substance P and CGRP in the DRG neurons may indicate a participation and function of Ang II in the regulation of nociception. In conclusion, these results suggest that Ang II may be produced locally in the neurons of rat and human DRG and act as a neurotransmitter.

Human mast cells express intracellular TRAIL

Recently we demonstrated that human mast cells (MC) express functional TRAIL death receptors. Here we assessed the expression of TRAIL on both mRNA and protein level in cord blood derived MC (CBMC) and HMC-1. The TRAIL release either spontaneous or induced by LPS, IFN-gamma and IgE-dependent activation, was evaluated as well. The protein location was restricted to the intracellular compartment in CBMC, but not in HMC-1. The intracellular TRAIL was not localized inside the granules. The treatment with IFN-gamma and LPS up-regulated intracellular TRAIL expression in CBMC, but did not induce its release. These in vitro data show that human MC can produce and express intracellular TRAIL whose location could not be altered by different stimuli.

Intracellular imaging of nanoparticles: is it an elemental mistake to believe what you see?

In order to understand how nanoparticles (NPs <100 nm) interact with cellular systems, potentially causing adverse effects, it is important to be able to detect and localize them within cells. Due to the small size of NPs, transmission electron microscopy (TEM) is an appropriate technique to use for visualizing NPs inside cells, since light microscopy fails to resolve them at a single particle level. However, the presence of other cellular and non-cellular nano-sized structures in TEM cell samples, which may resemble NPs in size, morphology and electron density, can obstruct the precise intracellular identification of NPs. Therefore, elemental analysis is recommended to confirm the presence of NPs inside the cell. The present study highlights the necessity to perform elemental analysis, specifically energy filtering TEM, to confirm intracellular NP localization using the example of quantum dots (QDs). Recently, QDs have gained increased attention due to their fluorescent characteristics, and possible applications for biomedical imaging have been suggested. Nevertheless, potential adverse effects cannot be excluded and some studies point to a correlation between intracellular particle localization and toxic effects. J774.A1 murine macrophage-like cells were exposed to NH2 polyethylene (PEG) QDs and elemental co-localization analysis of two elements present in the QDs (sulfur and cadmium) was performed on putative intracellular QDs with electron spectroscopic imaging (ESI). Both elements were shown on a single particle level and QDs were confirmed to be located inside intracellular vesicles. Nevertheless, ESI analysis showed that not all nano-sized structures, initially identified as QDs, were confirmed. This observation emphasizes the necessity to perform elemental analysis when investigating intracellular NP localization using TEM.

Fluorescent-magnetic hybrid nanoparticles induce a dose-dependent increase in proinflammatory response in lung cells in vitro correlated with intracellular localization

Iron-platinum nanoparticles embedded in a poly(methacrylic acid) (PMA) polymer shell and fluorescently labeled with the dye ATTO 590 (FePt-PMA-ATTO-2%) are investigated in terms of their intracellular localization in lung cells and potential to induce a proinflammatory response dependent on concentration and incubation time. A gold core coated with the same polymer shell (Au-PMA-ATTO-2%) is also included. Using laser scanning and electron microscopy techniques, it is shown that the FePt-PMA-ATTO-2% particles penetrate all three types of cell investigated but to a higher extent in macrophages and dendritic cells than epithelial cells. In both cell types of the defense system but not in epithelial cells, a particle-dose-dependent increase of the cytokine tumor necrosis factor alpha (TNFalpha) is found. By comparing the different nanoparticles and the mere polymer shell, it is shown that the cores combined with the shells are responsible for the induction of proinflammatory effects and not the shells alone. It is concluded that the uptake behavior and the proinflammatory response upon particle exposure are dependent on the time, cell type, and cell culture.

Juice of Bryophyllum pinnatum (Lam.) inhibits oxytocin-induced increase of the intracellular calcium concentration in human myometrial cells

http://www.ncbi.nlm.nih.gov/pubmed/20381326

Confocal imaging protocols for live/dead staining in three-dimensional carriers

In tissue engineering, a variety of methods are commonly used to evaluate survival of cells inside tissues or three-dimensional (3D) carriers. Among these methods confocal laser scanning microscopy opened accessibility of 3D tissue using live cell imaging into the tissue or 3D scaffolds. However, although this technique is ideally applied to 3D tissue or scaffolds with thickness up to several millimetres, this application is surprisingly rare and scans are often done on slices with thickness <20 μm. Here, we present novel protocols for the staining of 3D tissue (e.g. intervertebral disc tissue) and scaffolds, such as fibrin gels or alginate beads.

The influence of toothbrushing and coffee staining on different composite surface coatings

The aim of our study is to evaluate the performance of surface sealants and conventional polishing after ageing procedures. Eighty circular composite restorations were performed on extracted human molars. After standardised roughening, the restorations were either sealed with one of three surface sealants (Lasting Touch (LT), BisCover LV (BC), G-Coat Plus (GP) or a dentin adhesive Heliobond (HB)) or were manually polished with silicon polishers (MP) (n = 16). The average roughness (Ra) and colourimetric parameters (CP) (L*a*b*) were evaluated. The specimens underwent an artificial ageing process by thermocycling, staining (coffee) and abrasive (toothbrushing) procedures. After each ageing step, Ra and CP measurements were repeated. A qualitative surface analysis was performed with SEM. The differences between the test groups regarding Ra and CP values were analysed with nonparametric ANOVA analysis (α = 0.05). The lowest Ra values were achieved with HB. BC and GP resulted in Ra values below 0.2 μm (clinically relevant threshold), whereas LT and MP sometimes led to higher Ra values. LT showed a significantly higher discolouration after the first coffee staining, but this was normalised to the other groups after toothbrushing. The differences between the measurements and test groups for Ra and CP were statistically significant. However, the final colour difference showed no statistical difference among the five groups. SEM evaluation showed clear alterations after ageing in all coating groups. Surface sealants and dentin adhesives have the potential to reduce surface roughness but tend to debond over time. Surface sealants can only be recommended for polishing provisional restorations.

Drug target identification in intracellular and extracellular protozoan parasites

The increasing demand for novel anti-parasitic drugs due to resistance formation to well-established chemotherapeutically important compounds has increased the demands for a better understanding of the mechanism(s) of action of existing drugs and of drugs in development. While different approaches have been developed to identify the targets and thus mode of action of anti-parasitic compounds, it has become clear that many drugs act not only on one, but possibly several parasite molecules or even pathways. Ideally, these targets are not present in any cells of the host. In the case of apicomplexan parasites, the unique apicoplast, provides a suitable target for compounds binding to DNA or ribosomal RNA of prokaryotic origin. In the case of intracellular pathogens, a given drug might not only affect the pathogen by directly acting on parasite-associated targets, but also indirectly, by altering the host cell physiology. This in turn could affect the parasite development and lead to parasite death. In this review, we provide an overview of strategies for target identification, and present examples of selected drug targets, ranging from proteins to nucleic acids to intermediary metabolism.