Characterization of the Murine Myeloid Precursor Cell Line MuMac-E8

Starting point for the present work was the assumption that the cell line MuMac-E8 represents a murine cell population with stem cell properties. Preliminary studies already pointed to the expression of stem-cell associated markers and a self-regenerative potential of the cells. The cell line MuMac-E8 should be examined for their differential stage within stem cell hierarchy. MuMac-E8 cells were derived from a chimeric mouse model of arthritis. It could be shown that MuMac-E8 cells express mRNA of some genes associated with pluripotent stem cells (Nanog, Nucleostemin), of genes for hematopoietic markers (EPCR, Sca-1, CD11b, CD45), for the mesenchymal marker CD105 and of genes for the neural markers Pax-6 and Ezrin. In methylcellulose and May-Grünwald-Giemsa staining, hematopoietic colonies were obtained but the hematopoietic system of lethally irradiated mice could not be rescued. Osteogenic differentiation was not detectable. Thus, it became evident that MuMac-E8 represents not a stem cell line. However, MuMac-E8 cells expressed several myeloid surface markers (i.e. CD11b, F4/80, CD14, CD64), showed phagocytosis and is capable of producing nitric oxide. Thus, this cell line seems to be arrested an advanced stage of myeloid differentiation. Adherence data measured by impedance-based real-time cell analysis together with cell morphology data suggested that MuMac-E8 represents a new macrophage precursor cell line exhibiting weak adherence. This cell line is suitable as an in-vitro model for testing of macrophage functions. Moreover, it might be also useful for differentiation or reprogramming studies.     

Metabolic Signatures of Cultured Human Adipocytes from Metabolically Healthy versus Unhealthy Obese Individuals

Background and Aims

Among obese subjects, metabolically healthy and unhealthy obesity (MHO/MUHO) can be differentiated: the latter is characterized by whole-body insulin resistance, hepatic steatosis, and subclinical inflammation. Aim of this study was, to identify adipocyte-specific metabolic signatures and functional biomarkers for MHO versus MUHO.

Methods

10 insulin-resistant (IR) vs. 10 insulin-sensitive (IS) non-diabetic morbidly obese (BMI >40 kg/m²) Caucasians were matched for gender, age, BMI, and percentage of body fat. From subcutaneous fat biopsies, primary preadipocytes were isolated and differentiated to adipocytes in vitro. About 280 metabolites were investigated by a targeted metabolomic approach intracellularly, extracellularly, and in plasma.

Results/Interpretation

Among others, aspartate was reduced intracellularly to one third (p = 0.0039) in IR adipocytes, pointing to a relative depletion of citric acid cycle metabolites or reduced aspartate uptake in MUHO. Other amino acids, already known to correlate with diabetes and/or obesity, were identified to differ between MUHO''s and MHO''s adipocytes, namely glutamine, histidine, and spermidine. Most species of phosphatidylcholines (PCs) were lower in MUHO''s extracellular milieu, though simultaneously elevated intracellularly, e.g., PC aa C32∶3, pointing to increased PC synthesis and/or reduced PC release. Furthermore, altered arachidonic acid (AA) metabolism was found: 15(S)-HETE (15-hydroxy-eicosatetraenoic acid; 0 vs. 120pM; p = 0.0014), AA (1.5-fold; p = 0.0055) and docosahexaenoic acid (DHA, C22∶6; 2-fold; p = 0.0033) were higher in MUHO. This emphasizes a direct contribution of adipocytes to local adipose tissue inflammation. Elevated DHA, as an inhibitor of prostaglandin synthesis, might be a hint for counter-regulatory mechanisms in MUHO.

Conclusion/Interpretation

We identified adipocyte-inherent metabolic alterations discriminating between MHO and MUHO.     

Ceramide-1-phosphate blocks apoptosis through inhibition of acid sphingomyelinase in macrophages

It was reported previously that ceramide-1-phosphate (Cer-1-P) is mitogenic for fibroblasts (Gómez-Mu?oz, A., P. A. Duffy, A. Martin, L. O'Brien, H-S. Byun, R. Bittman, and D. N. Brindley. 1995. Mol. Pharmacol. 47: 883-889; Gómez-Mu?oz, A., L. M. Frago, L. Alvarez, and I. Varela-Nieto. 1997. Biochem. J. 325: 435-440). We now show that Cer-1-P prevents cell death in bone-marrow-derived macrophages (BMDMs) after withdrawal of macrophage colony-stimulating factor (M-CSF). Removal of M-CSF is known to induce apoptosis in these cells. Cer-1-P blocked activation of the caspase-9/caspase-3 pathway and prevented DNA fragmentation, indicating that the enhancement of cell survival was due to inhibition of apoptosis. M-CSF deprivation resulted in activation of acid sphingomyelinase (A-SMase), increased ceramide levels, and a decrease in intracellular Cer-1-P. Exogenously added Cer-1-P inhibited A-SMase in intact BMDMs at concentrations that also prevented apoptosis. Cer-1-P also inhibited A-SMase in cell homogenates, suggesting a possible direct physical interaction of Cer-1-P with the enzyme. In conclusion, these data demonstrate that Cer-1-P blocks apoptosis in BMDMs through inhibition of A-SMase, thereby reducing ceramide generation. This adds a new dimension to the understanding of the metabolic interrelationship of ceramides and Cer-1-P, and shows how altering the balance of intracellular levels of these mediators can affect cell survival.     

Flamingo regulates R8 axon-axon and axon-target interactions in the Drosophila visual system

Photoreceptors (R cells) in the Drosophila retina connect to targets in three distinct layers of the optic lobe of the brain: R1-R6 connect to the lamina, and R7 and R8 connect to distinct layers in the medulla. In each of these layers, R axon termini are arranged in evenly spaced topographic arrays. In a genetic screen for mutants with abnormal R cell connectivity, we recovered mutations in flamingo (fmi). fmi encodes a seven-transmembrane cadherin, previously shown to function in planar cell polarity and in dendritic patterning. Here, we show that fmi has two specific functions in R8 axon targeting: it facilitates competitive interactions between adjacent R8 axons to ensure their correct spacing, and it promotes the formation of stable connections between R8 axons and their target cells in the medulla. The former suggests a general role for Fmi in establishing nonoverlapping dendritic and axonal target fields. The latter, together with the finding that N-Cadherin has an analogous role in R7 axon-target interactions, points to a cadherin-based system for target layer specificity in the Drosophila visual system.     

Metal ions and prion diseases

Recent data demonstrate that transition metal ions such as copper not only bind the prion protein with high affinities, but also modify its biochemical properties. This has important consequences for the potential function of the protein in metal-ion transport or as an anti-oxidant molecule. In addition, this relationship between the prion protein and metal ions is likely to play a critical role in the physiopathology of prion diseases.     

Rapid alterations of cell cycle control proteins in human T lymphocytes in microgravity

ABSTRACT: In our study we aimed to identify rapidly reacting gravity-responsive mechanisms in mammalian cells in order to understand if and how altered gravity is translated into a cellular response. In a combination of experiments using "functional weightlessness" provided by 2D-clinostats and real microgravity provided by several parabolic flight campaigns and compared to in-flight-1g-controls, we identified rapid gravity-responsive reactions inside the cell cycle regulatory machinery of human T lymphocytes. In response to 2D clinorotation, we detected an enhanced expression of p21 Waf1/Cip1 protein within minutes, less cdc25C protein expression and enhanced Ser147-phosphorylation of cyclinB1 after CD3/CD28 stimulation. Additionally, during 2D clinorotation, Tyr-15-phosphorylation occurred later and was shorter than in the 1 g controls. In CD3/CD28-stimulated primary human T cells, mRNA expression of the cell cycle arrest protein p21 increased 4.1-fold after 20s real microgravity in primary CD4+ T cells and 2.9-fold in Jurkat T cells, compared to 1 g in-flight controls after CD3/CD28 stimulation. The histone acetyltransferase (HAT) inhibitor curcumin was able to abrogate microgravity-induced p21 mRNA expression, whereas expression was enhanced by a histone deacetylase (HDAC) inhibitor. Therefore, we suppose that cell cycle progression in human T lymphocytes requires Earth gravity and that the disturbed expression of cell cycle regulatory proteins could contribute to the breakdown of the human immune system in space.     

Antivascular therapy for multidrug-resistant ovarian tumors by macitentan, a dual endothelin receptor antagonist

Endothelin receptors (ETRs) are often overexpressed in ovarian tumors, which can be resistant to conventional therapies. Thus, we investigated whether blockage of the ETR pathways using the dual ETR antagonist macitentan combined with taxol or cisplatinum can produce therapy for orthotopically growing multidrug-resistant (MDR) human ovarian carcinoma. In several studies, nude mice were injected in the peritoneal cavity with HeyA8-MDR human ovarian cancer cells. Ten days later, mice were randomized to receive vehicle (saline), macitentan (oral, daily), taxol (intraperitoneal, weekly), cisplatinum (intraperitoneal, weekly), macitentan plus taxol, or macitentan plus cisplatinum. Moribund mice were killed, and tumors were collected, weighed, and prepared for immunohistochemical analysis. The HeyA8-MDR tumors did not respond to taxol, cisplatinum, or macitentan administered as single agents. In contrast, combination therapy with macitentan and taxol or macitentan and cisplatinum significantly decreased the tumor incidence and weight and significantly increased the survival of mice and their general condition. Multiple immunohistochemical analyses revealed that treatment with macitentan and macitentan plus taxol or cisplatinum inhibited the phosphorylation of ETRs, decreased the levels of pVEGFR2, pAkt, and pMAPK in tumor cells after 2 weeks of treatment and induced a first wave of apoptosis in tumor-associated endothelial cells followed by apoptosis in surrounding tumor cells. Our study shows that ovarian cancer cells, which express the endothelin axis and are multidrug resistant, are exquisitely sensitive to treatment with a dual ET antagonist and can be resensitized to both taxol and cisplatinum. This combined therapy led to a significant reduction in tumor weight.     

Crystal structure of Caulobacter crescentus polynucleotide phosphorylase reveals a mechanism of RNA substrate channelling and RNA degradosome assembly

Polynucleotide phosphorylase (PNPase) is an exoribonuclease that cleaves single-stranded RNA substrates with 3'-5' directionality and processive behaviour. Its ring-like, trimeric architecture creates a central channel where phosphorolytic active sites reside. One face of the ring is decorated with RNA-binding K-homology (KH) and S1 domains, but exactly how these domains help to direct the 3' end of single-stranded RNA substrates towards the active sites is an unsolved puzzle. Insight into this process is provided by our crystal structures of RNA-bound and apo Caulobacter crescentus PNPase. In the RNA-free form, the S1 domains adopt a 'splayed' conformation that may facilitate capture of RNA substrates. In the RNA-bound structure, the three KH domains collectively close upon the RNA and direct the 3' end towards a constricted aperture at the entrance of the central channel. The KH domains make non-equivalent interactions with the RNA, and there is a marked asymmetry within the catalytic core of the enzyme. On the basis of these data, we propose that structural non-equivalence, induced upon RNA binding, helps to channel substrate to the active sites through mechanical ratcheting. Structural and biochemical analyses also reveal the basis for PNPase association with RNase E in the multi-enzyme RNA degradosome assembly of the α-proteobacteria.     

Ketamine Decreases Resting State Functional Network Connectivity in Healthy Subjects: Implications for Antidepressant Drug Action

Increasing preclinical and clinical evidence underscores the strong and rapid antidepressant properties of the glutamate-modulating NMDA receptor antagonist ketamine. Targeting the glutamatergic system might thus provide a novel molecular strategy for antidepressant treatment. Since glutamate is the most abundant and major excitatory neurotransmitter in the brain, pathophysiological changes in glutamatergic signaling are likely to affect neurobehavioral plasticity, information processing and large-scale changes in functional brain connectivity underlying certain symptoms of major depressive disorder. Using resting state functional magnetic resonance imaging (rsfMRI), the „dorsal nexus “(DN) was recently identified as a bilateral dorsal medial prefrontal cortex region showing dramatically increased depression-associated functional connectivity with large portions of a cognitive control network (CCN), the default mode network (DMN), and a rostral affective network (AN). Hence, Sheline and colleagues (2010) proposed that reducing increased connectivity of the DN might play a critical role in reducing depression symptomatology and thus represent a potential therapy target for affective disorders. Here, using a randomized, placebo-controlled, double-blind, crossover rsfMRI challenge in healthy subjects we demonstrate that ketamine decreases functional connectivity of the DMN to the DN and to the pregenual anterior cingulate (PACC) and medioprefrontal cortex (MPFC) via its representative hub, the posterior cingulate cortex (PCC). These findings in healthy subjects may serve as a model to elucidate potential biomechanisms that are addressed by successful treatment of major depression. This notion is further supported by the temporal overlap of our observation of subacute functional network modulation after 24 hours with the peak of efficacy following an intravenous ketamine administration in treatment-resistant depression.