Einfluss von Zinkfingerprotein 580 (ZNF580) auf Matrixmetalloprotease-1 (MMP-1) in humanen Podozyten

Die dysregulierte Sekretion von Matrixmetalloproteasen (MMP) durch Podozyten scheint eine Schlüsselrolle für die Beeinträchtigung der Integrität der glomerulären Filtrationsbarriere und die Entwicklung einer Proteinurie im Rahmen eines chronischen Nierenversagens zu spielen. Zinkfingerprotein 580 (ZNF580) ist ein potenzieller Transkriptionsfaktor aus der Klasse der C2H2-Typ-Zinkfingerproteine, welcher in Endothelzellen eine Beteiligung an der MMP-Expression zeigte. Entsprechende Versuche in Podozyten wurden noch nicht durchgeführt. In dieser Arbeit soll in einer immortalisierten humanen Podozytenzelllinie der Einfluss von ZNF580 auf die Expression von MMP untersucht und daraus Rückschlüsse auf die molekulare Pathophysiologie chronischer Nierenerkrankungen gezogen werden. Die Lokalisation von ZNF580 in Podozyten wurde mit Immunfluoreszenzmikroskopie sowie Immunoblotting nach Zellfraktionierung untersucht. Mittels spezifischer ZNF580-esiRNA konnte in Podozyten ein ZNF580-Knockdown erreicht werden, die Zellen wurden im Anschluss systematisch auf Veränderungen der relativen mRNA-Konzentration verschiedener MMP analysiert. Weiterhin wurde der Einfluss des Zytokins IL-1β (Stimulus der MMP-Expression) sowie verschiedener MAPK auf ZNF580 und MMP in Podozyten untersucht. Veränderungen auf mRNA-Ebene wurden dabei stets mit quantitativer RT-PCR, Unterschiede auf Proteinebene mit-tels Western Blot analysiert. ZNF580 konnte im Zellkern von Podozyten lokalisiert werden. ZNF580-Knockdown mittels spezifischer esiRNA führte zu einem Anstieg der MMP-1-mRNA-Konzentration, während die mRNA-Konzentrationen aller anderen untersuchten MMP und TIMP (endogenen MMP-Inhibitoren) unverändert blieben. Durch Stimulation von Podozyten mit IL-1β konnte eine Induktion der MMP-1- sowie eine Suppression der ZNF580-Expression herbeigeführt werden. Die spezifische Inhibition verschiedener MAPK (p38, JNK sowie ERK) führte zu einer Abschwächung der MMP-1-Induktion nach IL-1β-Stimulation. ZNF580 scheint somit ein Repressor der MMP-1-Expression in Podozyten zu sein. Die Stimulation von Podozyten mit IL-1β führte also durch die Suppression von ZNF580 zu einem Wegfall dieser „Bremse“ und somit zu vermehrter MMP-1-Expression. Die Lokalisation von ZNF580 im Zellkern von Podozyten stärkt dabei die Hypothese, dass ZNF580 als Transkriptionsfaktor wirkt. Angesichts der mutmaßlichen Beteiligung von MMP-1 an der Entwicklung der Proteinurie bei chronischen Nierenerkrankungen ist ZNF580 daher als ein potenziell protektiver Faktor in der Pathophysiologie der Proteinurie anzusehen.A dysregulated expression and secretion of matrix metalloproteinases (MMPs) by podocytes seems to play a key role for the impairment of the integrity of the glomerular filtration barrier and the development of proteinuria in the context of chronic kidney failure. Zinc finger protein 580 (ZNF580), a putative C2H2 zinc finger transcription factor, participates in the regulation of MMP expression in endothelial cells. However, similar experiments have not yet been performed in podocytes. Therefore, the aim of the present thesis was to investigate possible relationships between ZNF580 and MMP expression in an immortalized human podocyte cell line and to draw conclusions about the molecular pathophysiology of chronic kidney diseases. The localization of ZNF580 in podocytes was investigated by immunofluorescence microscopy and immunoblotting after seperating nuclear and zytosolic protein fractions. ZNF580 knockdown in podocytes was achieved using specific ZNF580 esiRNA, the cells were subsequently systematically examined about changes in the relative mRNA concentrations of different MMPs. Furthermore, the impact of IL-1β (inflammatory cytokine and stimulus of MMP expression) and different MAPK on ZNF580 and MMP expression in podocytes was investigated. Changes at the mRNA level were analyzed using quantitative RT-PCR, differences at the protein level by western blot. ZNF580 was localized in the nucleus of podocytes. ZNF580 knockdown by specific ZNF580 esiRNA resulted in increased MMP-1 mRNA concentration, while the mRNA concentrations of other MMPs and TIMPs (endogenous inhibitors of MMPs) remained unchanged. Stimulating podocytes with IL-1β led to induction of MMP-1 and suppression of ZNF580 expression. Specific inhibition of different MAPK (p38, JNK and ERK) revealed attenuated induction of MMP-1 after stimulation of podocytes with IL-1β. Thus, ZNF580 seems to act as a repressor of the MMP-1 expression in podocytes. Stimulation of podocytes with IL-1β led to the suppression of ZNF580 so that this "brake" of the MMP-1 expression was attenuated. Consequently, an induction of MMP-1 was observed. The localization of ZNF580 in the nucleus of podocytes strengthens the hypothesis that ZNF580 operates as a transcription factor. Given the role of MMP-1 in the development of proteinuria ZNF580 appears to act as a protective factor in the pathophysiology of proteinuria

Sich informieren, sich inspirieren lassen, netzwerken und feiern : Bericht von der BiblioCon 2023 in Hannover

Vom 23.05.2023 bis zum 26.05.2023 fand die BiblioCon in Hannover statt. Matthias Finck und Ulrike Spree waren dabei und berichten über ihre persönlichen Eindrücke, u. a. mit Schilderungen zu Open-Source-Lösungen im Bibliotheksalltag, zu Wikimedians in Bibliotheken und zum Hands-On Lab „Das perfekte Praktikum“

Relation of nNOS isoforms to mitochondrial density and PGC-1alpha expression in striated muscles of mice

The expression of neuronal NO synthase (nNOS) alpha- and beta-isoforms in skeletal muscle is well documented but only little information is available about their regulation/functions. Using different mouse models, we now assessed whether the expression of nNOS-isoforms in muscle fibers is related to mitochondria content/activity and regulated by peroxisome proliferator-activated receptor gamma coactivator-1alpha (PGC-1alpha). Catalytic histochemistry revealed highest nNOS-concentrations to be present in type-2 oxidative muscle fibers. Differences in mitochondrial density between nNOS-KO-mice and WT-littermates established by morphometry after transmission electron microscopy were significant in the oxidative portion of the tibialis anterior muscle (TA) but not in rectus femoris muscle (RF) indicating an nNOS-dependent mitochondrial pool in TA. Quantitative immunoblotting displayed the nNOS alpha-isoform to preponderate in those striated muscles of C57BL/6-mice that comprise of many type-2 oxidative fibers, e.g. TA, while roughly even levels of the two nNOS-isoforms were expressed in those muscles that mainly consist of type-2 glycolytic fibers, e.g. RF. Differences in citrate synthase-activity in muscle homogenates between nNOS-KO-mice and WT-littermates were positively related to nNOS alpha-isoform levels. In transgenic-mice over-expressing muscular PGC-1alpha compared to WT-littermates, immunoblotting revealed a significant shift in nNOS-expression in favor of the alpha-isoform in six out of eight striated muscles (exceptions: soleus muscle and tongue) without consistent relationship to changes in the expression of mitochondrial markers. In summary, our study demonstrated the nNOS alpha-isoform expression to be related to mitochondrial content/activity and to be up-regulated by up-stream PGC-1alpha in striated muscles, particularly in those enriched with type-2 oxidative fibers implying a functional convergence of the two signaling systems in these fibers

Metamagnetic transition and a loss of magnetic hysteresis caused by electron trapping in monolayers of single-molecule magnet Tb2_{2}@C79_{79}N

Whereas bulk Tb$_{2}$@C$_{79}$N is a single-molecule magnet with broad hysteresis, its monolayers on different substrates show the prevalence of a non-magnetic ground state near zero magnetic field and a metamagnetic transition with the field increase. Realization of stable spin states in surface-supported magnetic molecules is crucial for their applications in molecular spintronics, memory storage or quantum information processing. In this work, we studied the surface magnetism of dimetallo-azafullerene Tb$_{2}$@C$_{79}$N, showing a broad magnetic hysteresis in a bulk form. Surprisingly, monolayers of Tb$_{2}$@C$_{79}$N exhibited a completely different behavior, with the prevalence of a ground state with antiferromagnetic coupling at low magnetic field and a metamagnetic transition in the magnetic field of 2.5–4 T. Monolayers of Tb$_{2}$@C$_{79}$N were deposited onto Cu(111) and Au(111) by evaporation in ultra-high vacuum conditions, and their topography and electronic structure were characterized by scanning tunneling microscopy and spectroscopy (STM/STS). X-ray photoelectron spectroscopy (XPS), in combination with DFT studies, revealed that the nitrogen atom of the azafullerene cage tends to avoid metallic surfaces. Magnetic properties of the (sub)monolayers were then studied by X-ray magnetic circular dichroism (XMCD) at the Tb-M$_{4,5}$ absorption edge. While in bulk powder samples Tb$_{2}$@C$_{79}$N behaves as a single-molecule magnet with ferromagnetically coupled magnetic moments and blocking of magnetization at 28 K, its monolayers exhibited a different ground state with antiferromagnetic coupling of Tb magnetic moments. To understand if this unexpected behavior is caused by a strong hybridization of fullerenes with metallic substrates, XMCD measurements were also performed for Tb2@C79N adsorbed on h-BN|Rh(111) and MgO|Ag(100). The co-existence of two forms of Tb$_{2}$@C$_{79}$N was found on these substrates as well, but magnetization curves showed narrow magnetic hysteresis detectable up to 25 K. The non-magnetic state of Tb$_{2}$@C$_{79}$N in monolayers is assigned to anionic Tb$_{2}$@C$_{79}$N− species with doubly-occupied Tb–Tb bonding orbital and antiferromagnetic coupling of the Tb moments. A charge transfer from the substrate or trapping of secondary electrons are discussed as a plausible origin of these species

Sich informieren, sich inspirieren lassen, netzwerken und feiern : Bericht von der BiblioCon 2023 in Hannover

Vom 23.05.2023 bis zum 26.05.2023 fand die BiblioCon in Hannover statt. Matthias Finck und Ulrike Spree waren dabei und berichten über ihre persönlichen Eindrücke, u. a. mit Schilderungen zu Open-Source-Lösungen im Bibliotheksalltag, zu Wikimedians in Bibliotheken und zum Hands-On Lab „Das perfekte Praktikum“.BiblioCon took place from 23.05.2023 to 26.05.2023 in Hannover. Matthias Finck and Ulrike Spree were there and report their personal impressions, including descriptions of open source solutions in everyday library work, wiki medians in libraries and the hands-on lab „The Perfect Internship“.AlternativeReviewe

Metamagnetic transition and a loss of magnetic hysteresis caused by electron trapping in monolayers of single-molecule magnet Tb2@C79N

Realization of stable spin states in surface-supported magnetic molecules is crucial for their applications in molecular spintronics, memory storage or quantum information processing. In this work, we studied the surface magnetism of dimetallo-azafullerene Tb2@C79N, showing a broad magnetic hysteresis in a bulk form. Surprisingly, monolayers of Tb2@C79N exhibited a completely different behavior, with the prevalence of a ground state with antiferromagnetic coupling at low magnetic field and a metamagnetic transition in the magnetic field of 2.5-4 T. Monolayers of Tb2@C79N were deposited onto Cu(111) and Au(111) by evaporation in ultra-high vacuum conditions, and their topography and electronic structure were characterized by scanning tunneling microscopy and spectroscopy (STM/STS). X-ray photoelectron spectroscopy (XPS), in combination with DFT studies, revealed that the nitrogen atom of the azafullerene cage tends to avoid metallic surfaces. Magnetic properties of the (sub)monolayers were then studied by X-ray magnetic circular dichroism (XMCD) at the Tb-M4,5 absorption edge. While in bulk powder samples Tb2@C79N behaves as a single-molecule magnet with ferromagnetically coupled magnetic moments and blocking of magnetization at 28 K, its monolayers exhibited a different ground state with antiferromagnetic coupling of Tb magnetic moments. To understand if this unexpected behavior is caused by a strong hybridization of fullerenes with metallic substrates, XMCD measurements were also performed for Tb2@C79N adsorbed on h-BN|Rh(111) and MgO|Ag(100). The co-existence of two forms of Tb2@C79N was found on these substrates as well, but magnetization curves showed narrow magnetic hysteresis detectable up to 25 K. The non-magnetic state of Tb2@C79N in monolayers is assigned to anionic Tb2@C79N− species with doubly-occupied Tb-Tb bonding orbital and antiferromagnetic coupling of the Tb moments. A charge transfer from the substrate or trapping of secondary electrons are discussed as a plausible origin of these species