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Hormone stellen eine wichtige Ursache f��r geschlechtsspezifische Unterschiede bei Herz-Kreislauferkrankungen und Erkrankungen des Immunsystems dar. Daneben werden aber auch zunehmend intrinsische geschlechtsspezifische Unterschiede auf zellul��rer Ebene diskutiert. So wurde gezeigt, dass weibliche Zellen eine st��rkere Expression immunrelevanter Gene aufweisen als m��nnliche. Frauen besitzen gegen��ber M��nnern ein aktiveres Immunsystem, was auch dazu f��hrt, dass Frauen h��ufiger von Erkrankungen des Immunsystems betroffen sind. Zytokine und Chemokine vermitteln zahlreiche Prozesse immunbedingter und entz��ndlicher Erkrankungen. Daher sollte in dieser Arbeit ��berpr��ft werden, ob es geschlechtsspezifische Unterschiede in intrazellul��ren und sezernierten Zytokinen und Chemokinen bei Endothelzellen der Nabelschnur (HUVEC) gibt. Durch die Verwendung von HUVEC-Zellen k��nnen weitgehend hormonunabh��ngige geschlechtsspezifische Unterschiede untersucht werden. Mittels Zytokin- und Chemokinarrays konnten in dieser Arbeit geschlechtsspezifische Unterschiede nachgewiesen werden. Weibliche HUVEC sezernieren im Vergleich zu m��nnlichen Zellen weniger Zytokine und Chemokine in Zellkultur��berst��nde. So wurden h��here Konzentrationen von IL-8 und MCP-1 in Zellkultur��berst��nden m��nnlicher unbehandelter Zellen gemessen. Auch nach VEGF-Behandlung wurden h��here Konzentrationen von IL-8, MCP-1, Midkine, CXCL 16, IL-6 und C5/C5a in Zellkultur��berst��nden m��nnlicher HUVEC nachgewiesen. Im Gegensatz dazu wurden intrazellul��r mehr Zytokine und Chemokine (IL-8, GRO-��, Midkine, MIF, Serpin E1, IL-6, sICAM-1 und IL-1ra) in weiblichen unbehandelten HUVEC detektiert. Damit scheint es geschlechtsspezifische Unterschiede vor allem im Verh��ltnis sezernierter zu intrazellul��ren Zytokinen und Chemokinen zu geben. Die vorliegenden Ergebnisse verdeutlichen, dass Zellen intrinsische geschlechtsspezifische Unterschiede aufweisen und unterstreichen die Notwendigkeit das Geschlecht der Zellen zu ber��cksichtigen., Hormones represent an important cause for sex-specific differences in cardiovascular and of diseases of the immune system. In addition, intrinsic sex differences at the cellular level are increasingly being discussed. It has been shown that female cells have a stronger expression of immune-related genes than male cells. Women possess a more active immune system compared to men, which manifests itself in the fact that women are more frequently affected by diseases of the immune system. Cytokines and chemokines mediate numerous processes of immune-related and inflammatory diseases. Therefore, this work aimed to investigate whether there are sex-specific differences in intracellular and secreted cytokines and chemokines in human umbilical cord endothelial cells (HUVEC). By using HUVEC cells as an established model in basic research, largely hormone-independent sex differences can be investigated. Cytokine and chemokine arrays have been used in this work to detect sex-specific differences. Female HUVEC secrete fewer cytokines and chemokines in cell culture supernatants compared with male cells. Thus, higher levels of IL-8 and MCP-1 were measured in cell culture supernatants of untreated male cells. Higher concentrations of IL-8, MCP-1, midkine, CXCL 16, IL-6, and C5/C5a were also detected in cell culture supernatants of male HUVEC after VEGF treatment. In contrast, more cytokines and chemokines (IL-8, GRO-��, midkine, MIF, serpin E1, IL-6, sICAM-1, and IL-1ra) were detected intracellularly in untreated female HUVEC. Thus, it has been shown that sex-specific differences exist in the ratio of secreted to intracellular cytokines and chemokines. The present results highlight that cells exhibit intrinsic sex differences and emphasize the need to consider the sex of the cells in basic research.

The ability of cells to sense and respond to extracellular forces is critical for cellular and tissue homeostasis. Tension or compression act on our body ubiquitously and cells respond to such mechanical cues by producing intracellular forces on their own. In this article, we briefly highlight the cellular and physical basis driving these phenomena and discuss our recent technical advance to stimulate and monitor the cellular mechanoresponse on a molecular scale.

Biospektrum : Mitteilungsorgan der Gesellschaft für Biochemie und Molekularbiologie (GBM), der Vereinigung für Allgemeine und Angewandte Mikrobiologie (VAAM), der Gesellschaft für Genetik (GfG), der Gesellschaft für Entwicklungsbiologie (GfE) und der Deutschen Gesellschaft für Experimentelle und Klinische Pharmakologie und Toxikologie (DGPT) 26(5), 478-481 (2020). doi:10.1007/s12268-020-1434-y, Published by Spektrum, Heidelberg

The ADAMTS9 rs4607103 C allele is one of the few gene variants proposed to increase the risk of type 2 diabetes through an impairment of insulin sensitivity. We show that the variant is associated with increased expression of the secreted ADAMTS9 and decreased insulin sensitivity and signaling in human skeletal muscle. In line with this, mice lacking Adamts9 selectively in skeletal muscle have improved insulin sensitivity. The molecular link between ADAMTS9 and insulin signaling was characterized further in a model where ADAMTS9 was overexpressed in skeletal muscle. This selective overexpression resulted in decreased insulin signaling presumably mediated through alterations of the integrin β1 signaling pathway and disruption of the intracellular cytoskeletal organization. Furthermore, this led to impaired mitochondrial function in mouse muscle—an observation found to be of translational character because humans carrying the ADAMTS9 risk allele have decreased expression of mitochondrial markers. Finally, we found that the link between ADAMTS9 overexpression and impaired insulin signaling could be due to accumulation of harmful lipid intermediates. Our findings contribute to the understanding of the molecular mechanisms underlying insulin resistance and type 2 diabetes and point to inhibition of ADAMTS9 as a potential novel mode of treating insulin resistance.

In view of our more comprehensive understanding with respect to intracellular pathways and their regulation, a vast number of interesting drug targets appear to be in a space that can be addressed neither by small molecules nor by biologics. Especially, interference with intracellular protein–protein interactions, if successful, seems to offer considerable opportunities to expand the application of peptides as therapeutics, in particular in the field of oncology. This review focuses on requirements for the development of peptide therapeutics aiming at intracellular targets. In addition, an outlook for developments in this field based on recent examples for peptides active in cellular assays, highlighting key requirement to assess permeability, is provided.

This review describes a new method for quantitative measurement and spatial imaging of microviscosity within individual domains of live cells. The method is based on fluorescence detection from small synthetic molecules termed 'molecular rotors', which are characterised by a strong response of fluorescence lifetimes or spectra to the viscosity of their immediate environment. We have demonstrated that the quantitative determination of viscosity is possible using lifetime-based molecular rotors and ratiometric molecular rotors. The ratiometric imaging of viscosity benefits from a very fast signal acquisition. We have illustrated this advantage by monitoring changes in intracellular viscosity during photodynamic therapy, which is a clinically utilised modality for the treatment of neoplastic disease.

Microbial biofilms show properties not predictable from those of single cells, e.g., the formation of a matrix of extracellular polymeric substances (EPS) with high cell densities, intense intercellular communication, concentration gradients, and structural heterogeneity. The result: a wide variety of habitats and the formation of multispecies microbial consortia. Matrix formation and dispersion, expression of virulence factors, tole — rance to antibiotics, and disinfectants are regulated. These features can be regarded as “emergent properties” of biofilms.

In macrophages, HIV-1 accumulates in intracellular vesicles designated virus-containing compartments (VCCs). These might play an important role in the constitution of macrophages as viral reservoirs and allow HIV-1 to evade the immune system by sequestration in an internal niche, which is difficult to access from the exterior. However, until now, evidence of whether internal virus accumulations are protected from the host's humoral immune response is still lacking. In order to be able to study the formation and antibody accessibility of VCCs, we generated HIV-1 with green fluorescent protein (GFP)-tagged Gag replicating in primary macrophages. Live-cell observations revealed faint initial cytosolic Gag expression and subsequent large intracellular Gag accumulations which stayed stable over days. Taking advantage of the opportunity to study the accessibility of intracellular VCCs via the cell surface, we demonstrate that macrophage internal HIV-1-containing compartments cannot be targeted by neutralizing antibodies. Furthermore, HIV-1 was efficiently transferred from antibody-treated macrophages to T cells. Three-dimensional reconstruction of electron microscopic slices revealed that Gag accumulations correspond to viral particles within enclosed compartments and convoluted membranes. Thus, although some VCCs were connected to the plasma membrane, the complex membrane architecture of the HIV-1-containing compartment might shield viral particles from neutralizing antibodies. In sum, our study provides evidence that HIV-1 is sequestered into a macrophage internal membranous web, posing an obstacle for the elimination of this viral reservoir.

The cisplatin adduct of vitamin B12, cis-[PtCl(NH3)2B12]+ (1) reacts with dansyl-imidazole to form cis-[Pt(dansyl-imidazole)(NH3)2B12] (2). In a spectroelectrochemical assay, Co(III) in 2 was reduced to Co(II) under concomitant release of the Pt(II) complex. Since similar reduction occurs in the intracellular space, 2 can be used for studying cell uptake monitored by fluorescent microscopy. The insertion of a fluorescent marker into the platinum moiety will also facilitate the isolation of the platinum complex released from B12 and therefore to characterize its chemical authenticity.

Die Art Listeria monocytogenes ist in erster Linie durch ihr pathogenes Potenzial im Menschen charakterisiert. Ihre nur fakultativ intrazelluläre Lebensweise ermöglicht es den Listerien aber auch, in diversen Habitaten in der Umwelt zu persistieren. In der vorliegenden Arbeit wurde das Überleben von L. monocytogenes in Protozoen der Gattung Acanthamoeba untersucht. Ein Assay, der es erlaubt, Infektionsstudien über mehrere Wochen durchzuführen, wurde entwickelt, für die Art L. monocytogenes optimiert und mit zahlreichen Kontrollen abgesichert. Interessanterweise konnten alle untersuchten zur Verfügung stehenden Virulenzgenmutanten ebenso wie Vertreter aus allen drei L. monocytogenes-Genotypen in Acanthamöben überleben. Das Überleben in Umweltprotozoen ist also ein Charakteristikum, das nicht von der Aktivität bekannter Virulenzgene abhängt. Durch Transmissionselektronenmikroskopie infizierter Amöbenzellen konnte gezeigt werden, dass sich L. monocytogenes-Zellen in Trophozoiten, dem teilungsfähigen und stoffwechselaktiven Stadium der Acanthamöben, im Gegensatz zur menschlichen Wirtszelle in Vakuolen befinden, während mittels konfokaler Laserscanning-Mikroskopie die Persistenz der Listerien in Amöbencysten in der Cystenwand nachgewiesen wurde. Der Verlust listerieller Virulenzgene wird in Umweltisolaten, die unter Umständen über längere Zeit nicht mit Wirtszellen in Kontakt waren, nicht beobachtet. Auch wenn für die Persistenz in Acanthamöben diese Virulenzfaktoren nicht benötigt werden, so könnte doch eine Erklärung darin liegen, dass diese Gene für das Überleben in weiteren niederen Eukaryonten benötigt werden. In dieser Arbeit konnte die Virulenzgenexpression in L. monocytogenes auch bei niedrigen Temperaturen durch einen RT-PCR-Ansatz und die damit verbundene Amplifikation von mRNS des listeriellen Virulenzgens plcB gezeigt werden. Zudem wurde mit Hilfe von sogenannten Enclosures die Persistenz von L. monocytogenes in der Umwelt hinsichtlich stammspezifischer Unterschiede der drei L. monocytogenes-Genotypen untersucht. In diesem Zusammenhang konnten Vertreter des Genotyps III, die sich durch eine niedrige Virulenz in Säugerzellen auszeichnen, durch ihre hohe Umweltpersistenz charakterisiert werden. Im Gegensatz dazu sind einige hochvirulente Vertreter der Art L. monocytogenes aus den Genotypen I und II nach längeren Inkubationsperioden nicht mehr in der Umwelt detektierbar. Diese Stämme sind demnach möglicherweise stärker auf den Menschen als Wirt spezialisiert und können in anderen Habitaten nicht gegen niedriger virulente Stämme konkurrieren. In einem weiteren Versuch wurden in dieser Arbeit Genotyp-spezifische Unterschiede innerhalb der Art L. monocytogenes hinsichtlich ihrer Virulenz in Säugerzelllinien untersucht. Interessanterweise sind Stämme des Genotyps III annähernd avirulent gegenüber menschlichen Darmendothelzellen, wohingegen in allen untersuchten Stämmen die gleiche Virulenz für Hepatocyten und Makrophagen bestimmt werden konnte. Die Attenuation von Vertretern des Genotyps III könnte also bereits zu Beginn einer Infektion des Wirtes in vivo auf eine beeinträchtige Infektiosität im Darmepithel, mit dem die Listerien durch die Aufnahme kontaminierter Nahrung in Kontakt kommen, zurückzuführen sein. Die untersuchten Stämme sind zwar attenuiert im intrazellulären Überleben in den entsprechenden Endothelzellen, können aber ebenso wie Vertreter aus den Genotypen I und II gleichermaßen effektiv an diese Wirtszellen adhärieren. Dies wurde durch Untersuchungen mit dem die Aktinumlagerung verhindernden, toxischen Agens Cytochalasin D gezeigt. The species Listeria monocytogenes is primarily characterized by its pathogenic potential in human beings. Its only facultatively intracellular lifestyle allows Listeriae to persist also in numerous habitats in the environment. In this thesis, the survival of L. monocytogenes within protozoa of the genus Acanthamoeba was investigated. For this purpose, an assay was developed to perform infection studies in a timeframe of several weeks. This assay was optimized for the species L. monocytogenes and collateralized by numerous control experiments. Interestingly, all virulence gene mutants investigated, as well as representatives from all three L. monocytogenes genotypes were able to survive within acanthamoebae. The survival in environmental protozoa is therefore not dependent on the activity of known virulence genes. Transmission electron microscopical studies of infected amoebae showed L. monocytogenes cells within vacuoles in the trophozoites, the metabolically active life stage of acanthamoebae. This is in contrast to mammalian host cells, where the bacteria lyse the phagosome and are present within the cytosol. Confocal laser scanning microscopy proved the existence of L. monocytogenes within the amoebal cyst wall. The loss of listerial virulence genes is seldomly seen even in environmental isolates which probably do not thrive within mammalian host cells for extended time periods. Even if virulence factors are not required for the persistence of L. monocytogenes in acanthamoebae, one explanation for this phenomenon could be the requirement of those genes for the survival in other lower eucaryotes. In this thesis, the expression of virulence genes in L. monocytogenes at lower temperatures could be shown by the amplification of the mRNA of the PrfA controlled listerial virulence gene plcB using RT-PCR. The persistence of L. monocytogenes in the environment was furthermore investigated with regards to strain specific differences between the three L. monocytogenes genotypes. Strains of genotype III, which feature low virulence in mammalian cells, were characterized by a high persistence in the environment. In contrast, highly virulent strains from genotypes I and II are no longer detectable in the environment after extended incubation times. Those strains are possibly more specializing in human / mammalian hosts and cannot compete against strains with lower virulence. In a further experiment, genotype specific differences within the species L. monocytogenes were investigated in terms of their virulence in mammalian cell lines. Interestingly, strains of genotype III are nearly avirulent against human endothelial Caco-2 cells, whereas all three genotypes were characterized by the same virulence against hepatocytes and macrophages. The attenuated virulence of genotype III strains could therefore possibly result from their impaired ability to infect the gut endothelium at this very early stage of the infectious intracellular cycle within the host. Though all genotype III strains investigated are attenuated in intracellular survival within the endothelial cells, they are able to attach to those host cells as efficiently as genotype I and II strains. This has been shown by experiments using Cytochalasin D, which inhibits actin rearrangement in the host cell.

Formation of tumor cell adhesion and following extravasation are limiting steps in organ specific metastasis. Focal Adhesion Kinase (FAK) plays a pivotal role in the intracellular signaling during cell adhesion and migration, which can be modulated by shear forces acting on circulating tumor cells. The intrinsic FAK inhibitor FRNK (FAK-related-non-kinase) was overexpressed in HEP G2 and HT-29 cells to investigate the role of FAK during early steps of metastasis. Static adhesion was not influenced by FRNK expression, whereas dynamic adhesion under flow conditions was dramatically reduced, apparent by significant reduced dynamic adhesion rates DAR (FRNK+: 12 ± 9 cells vs. FRNK-: 32 ± 10 cells; p 80%, p < 0.001) and complete inhibition of migration of the cells to the parenchyma. Our results indicate that FAK is involved in stabilization of cell adhesion on extracellular matrix in vitro and in vivo, which allows circulating tumor cells to resist the shear forces. This kinase is therefore involved in the regulation of rate limiting steps in distant metastasis formation.

During regeneration, retinal ganglion cell axons in fish upregulate a cell surface protein that is recognized by the monoclonal antibody (mAB) M802. M802 antigen appeared to be linked to the intracellular, membrane-associated lipid raft/microdomain proteins reggie-1 and reggie-2 that were previously shown to be reexpressed in axon-regenerating neurons [Development 124 (1997), 577]. Here, we report the isolation of the M802 antigen and its identification as the teleost homolog of mammalian Thy-1. Fish Thy-1 is detected in the same detergent-insoluble lipid raft fractions from a fibroblast cell line and from axon regenerating retinae as reggie-1 and 2. Importantly, mAB M802 coimmunoprecipitates reggie-1 and 2 from this lipid raft fraction, implying that fish Thy-1 and reggies interact. This correlates with their colocalization in growing cell processes after M802 antigen/Thy-1 activation with mAB M802. These findings suggest a role of clustered M802 antigen/Thy-1 in reggie raft microdomains for cell growth and axon regeneration.

Carboplatin (CBDCA) and the pyrimidine aglycone divicine displayed cytotoxic effects on murine erythroleukemic cells (MELC), with ID50 values of 158 and 37 μM, respectively. Combination of CBDCA and divicine, at a 2:1 ratio, increased cytotoxicity considerably. Under specific conditions of time schedule of administration, the association of CBDCA and divicine resulted in a clear synergistic activity. Alkaline elution studies on both unirradiated and γ-irradiated MELC demonstrated opposite patterns of DNA damage with the two molecules. Thus, CBDCA elicited DNA interstrand crosslinks (ISC), while divicine resulted in DNA single strand breaks (SSB). Association of both molecules led in the unirradiated cells to a higher SSB frequency than recorded with divicine alone. Accordingly, intracellular activation of CDBCA by redox cycling of divicine seems not to be involved. Rather, intracellular platinum appears to enhance cytotoxicity of divicine.

Summary The application of the degradation procedure for Gram-negative bacteria according to Bewick and Lo to Pasteurella multocida indicates that the obvious localization of the toxin is in the periplasm. The stability of the outer membrane and of the substances adhering to it is essential for the release of the toxin. The production of the toxin clearly depend on the media used.

A major portion of the lysosomal proteinases responsible for intracellular degradation of phagocytized proteins are cysteine proteinases, the “acid” cathepsins B, H, L, and S (see [1] for review). As we have shown previously [2, 3], increased cysteine proteinase activity is found in blood plasma of polytraumatized and septic patients as well as in local inflammatory secretions such as bronchoalveolar lavage fluid and peritonitis exudate. Most of this activity is due to cathepsin B, which is relatively stable at neutral pH (half-life about 30 min at pH 7.4) and is protected in the form of reversible complexes with its endogenenous protein inhibitors (stefins, cystatins, and kininogens). Here we report some evidence for the role of cathepsin B as a marker of macrophage activation and of lysosomal cysteine proteinases as potential nonspecific mediators of inflammation.

The effects of several amphipathic peptides on HIV-1 production in persistently infected cells are described. Melittin, a 26 amino acid α-helical amphipathic peptide, reduces HIV-1 production dose-dependently, whereas other amphipathic peptides do not. Six melittin derivatives which retain the α-helical portion have similar effects as melittin. The reduction of viral infectivity is not due to an effect of melittin on the virus particles but to an intracellular action of the peptide, which is readily taken up into cells, as shown by quantitative ELISA. Western blots of cells from melittin-treated cultures suggest that the processing of the gag/pol precursor is impaired.