A Natural Combination Extract of Viscum album L. Containing Both Triterpene Acids and Lectins Is Highly Effective against AML In Vivo

Aqueous Viscum album L. extracts are widely used in complementary cancer medicine. Hydrophobic triterpene acids also possess anti-cancer properties, but due to their low solubility they do not occur in significant amounts in aqueous extracts. Using cyclodextrins we solubilised mistletoe triterpenes (mainly oleanolic acid) and investigated the effect of a mistletoe whole plant extract on human acute myeloid leukaemia cells in vitro, ex vivo and in vivo. Single Viscum album L. extracts containing only solubilised triterpene acids (TT) or lectins (viscum) inhibited cell proliferation and induced apoptosis in a dose-dependent manner in vitro and ex vivo. The combination of viscum and TT extracts (viscumTT) enhanced the induction of apoptosis synergistically. The experiments demonstrated that all three extracts are able to induce apoptosis via caspase-8 and -9 dependent pathways with down-regulation of members of the inhibitor of apoptosis and Bcl-2 families of proteins. Finally, the acute myeloid leukaemia mouse model experiment confirmed the therapeutic effectiveness of viscumTT-treatment resulting in significant tumour weight reduction, comparable to the effect in cytarabine-treated mice. These results suggest that the combination viscumTT may have a potential therapeutic value for the treatment AML.     

9-Methyl-β-carboline-induced cognitive enhancement is associated with elevated hippocampal dopamine levels and dendritic and synaptic proliferation

β-Carbolines (BCs) belong to the heterogenous family of carbolines, which have been found exogenously, that is, in various fruits, meats, tobacco smoke, alcohol and coffee, but also endogenously, that is, blood, brain and CSF. These exogenous and endogenous BCs and some of their metabolites can exert neurotoxic effects, however, an unexpected stimulatory effect of 9-methyl-β-carboline (9-me-BC) on dopaminergic neurons in primary mesencephalic cultures was recently discovered. The aim of the present study was to extend our knowledge on the stimulatory effects of 9-me-BC and to test the hypothesis that 9-me-BC may act as a cognitive enhancer. We found that 10 days (but not 5 days) of pharmacological treatment with 9-me-BC (i) improves spatial learning in the radial maze, (ii) elevates dopamine levels in the hippocampal formation, and (iii) results after 10 days of treatment in elongated, more complex dendritic trees and higher spine numbers on granule neurons in the dentate gyrus of 9-me-BC-treated rats. Our results demonstrate that beyond its neuroprotective/neurorestorative and anti-inflammatory effects, 9-me-BC acts as a cognitive enhancer in a hippocampus-dependent task, and that the behavioral effects may be associated with a stimulatory impact on hippocampal dopamine levels and dendritic and synaptic proliferation.     

The Orphan G Protein-coupled Receptor GPR17 Negatively Regulates Oligodendrocyte Differentiation via Gαi/o and Its Downstream Effector Molecules

Recent studies have recognized G protein-coupled receptors as important regulators of oligodendrocyte development. GPR17, in particular, is an orphan G protein-coupled receptor that has been identified as oligodendroglial maturation inhibitor because its stimulation arrests primary mouse oligodendrocytes at a less differentiated stage. However, the intracellular signaling effectors transducing its activation remain poorly understood. Here, we use Oli-neu cells, an immortalized cell line derived from primary murine oligodendrocytes, and primary rat oligodendrocyte cultures as model systems to identify molecular targets that link cell surface GPR17 to oligodendrocyte maturation blockade. We demonstrate that stimulation of GPR17 by the small molecule agonist MDL29,951 (2-carboxy-4,6-dichloro-1H-indole-3-propionic acid) decreases myelin basic protein expression levels mainly by triggering the Gα_i/o signaling pathway, which in turn leads to reduced activity of the downstream cascade adenylyl cyclase-cAMP-PKA-cAMP response element-binding protein (CREB). In addition, we show that GPR17 activation also diminishes myelin basic protein abundance by lessening stimulation of the exchange protein directly activated by cAMP (EPAC), thus uncovering a previously unrecognized role for EPAC to regulate oligodendrocyte differentiation. Together, our data establish PKA and EPAC as key downstream effectors of GPR17 that inhibit oligodendrocyte maturation. We envisage that treatments augmenting PKA and/or EPAC activity represent a beneficial approach for therapeutic enhancement of remyelination in those demyelinating diseases where GPR17 is highly expressed, such as multiple sclerosis.     

Effect of cold environment on skeletal muscle mitochondria in growing rats

Summary Growing rats (4 weeks old) were kept for 3 weeks at 11° C and 24° C respectively. The cold-adapted animals showed a significantly higher oxygen consumption (64%). Volume density of subsarcolemmal and interfibrillar mitochondria as well as volume density of fat droplets were estimated in M. soleus and the diaphragm of both groups. In cold-adapted animals, the total volume of mitochondria was significantly increased by 24% in diaphragm and 37% in M. soleus. The volume of subsarcolemmal mitochondria was almost doubled in each muscle, but the volume of interfibrillar mitochondria did not change significantly. The surface of the inner mitochondrial membranes per unit volume of mitochondrion in M. soleus was significantly increased both in interfibrillar and subsarcolemmal mitochondria, whereas the surface of the outer mitochondrial membranes per unit volume of mitochondrion was increased only in the subsarcolemmal mitochondria. The volume of fat droplets in the diaphragm and M. soleus of cold adapted animals increased significantly by 62% and 150% respectively.     

A novel oxylipin-associated ‘ghosting’ phenomenon in yeast flocculation

Research on the distribution of oxylipins (3-hydroxy fatty acids) in flocculant strains of the yeast Saccharomyces cerevisiae led to the uncovering of a novel ghosting phenomenon observed during assumed lectin-mediated aggregation. We found that intracellular oxylipin-containing osmiophilic layers migrate through yeast cell walls in a ghostlike fashion without visually affecting the cell wall structure or the layers. This migration resulted in the binding of these layers to cell walls of adjacent cells. Consequently, ghosting seems a prerequisite for flocculation to occur. However, ghosting alone may not be sufficient to ensure flocculation.     

A Mammalian Cell Based FACS-Panning Platform for the Selection of HIV-1 Envelopes for Vaccine Development

An increasing number of broadly neutralizing monoclonal antibodies (bnMAb) against the HIV-1 envelope (Env) protein has been discovered recently. Despite this progress, vaccination efforts with the aim to re-elicit bnMAbs that provide protective immunity have failed so far. Herein, we describe the development of a mammalian cell based FACS-panning method in which bnMAbs are used as tools to select surface-exposed envelope variants according to their binding affinity. For that purpose, an HIV-1 derived lentiviral vector was developed to infect HEK293T cells at low multiplicity of infection (MOI) in order to link Env phenotype and genotype. For proof of principle, a gp145 Env model-library was established in which the complete V3 domain was substituted by five strain specific V3 loop sequences with known binding affinities to nMAb 447-52D, respectively. Env genes were recovered from selected cells by PCR, subcloned into a lentiviral vector (i) to determine and quantify the enrichment nMAb binders and (ii) to generate a new batch of transduction competent particles. After 2 selection cycles the Env variant with highest affinity was enriched 20-fold and represented 80% of the remaining Env population. Exploiting the recently described bnMAbs, this procedure might prove useful in selecting Env proteins from large Env libraries with the potential to elicit bnMAbs when used as vaccine candidates.     

The Thermosensitive Potassium Channel TREK-1 Contributes to Coolness-Evoked Responses of Grueneberg Ganglion Neurons

Neurons of the Grueneberg ganglion (GG) residing in the vestibule of the murine nose are activated by cool ambient temperatures. Activation of thermosensory neurons is usually mediated by thermosensitive ion channels of the transient receptor potential (TRP) family. However, there is no evidence for the expression of thermo-TRPs in the GG, suggesting that GG neurons utilize distinct mechanisms for their responsiveness to cool temperatures. In search for proteins that render GG neurons responsive to coolness, we have investigated whether TREK/TRAAK channels may play a role; in heterologous expression systems, these potassium channels have been previously found to close upon exposure to coolness, leading to a membrane depolarization. The results of the present study indicate that the thermosensitive potassium channel TREK-1 is expressed in those GG neurons that are responsive to cool temperatures. Studies analyzing TREK-deficient mice revealed that coolness-evoked responses of GG neurons were clearly attenuated in these animals compared with wild-type conspecifics. These data suggest that TREK-1 channels significantly contribute to the responsiveness of GG neurons to cool temperatures, further supporting the concept that TREK channels serve as thermoreceptors in sensory cells. Moreover, the present findings provide the first evidence of how thermosensory GG neurons are activated by given temperature stimuli in the absence of thermo-TRPs.     

Selectins Mediate Small Cell Lung Cancer Systemic Metastasis

Metastasis formation is the major reason for the extremely poor prognosis in small cell lung cancer (SCLC) patients. The molecular interaction partners regulating metastasis formation in SCLC are largely unidentified, however, from other tumor entities it is known that tumor cells use the adhesion molecules of the leukocyte adhesion cascade to attach to the endothelium at the site of the future metastasis. Using the human OH-1 SCLC line as a model, we found that these cells expressed E- and P-selectin binding sites, which could be in part attributed to the selectin binding carbohydrate motif sialyl Lewis A. In addition, protein backbones known to carry these glycotopes in other cell lines including PSGL-1, CD44 and CEA could be detected in in vitro and in vivo grown OH1 SCLC cells. By intravital microscopy of murine mesenterial vasculature we could capture SCLC cells while rolling along vessel walls demonstrating that SCLC cells mimic leukocyte rolling behavior in terms of selectin and selectin ligand interaction in vivo indicating that this mechanism might indeed be important for SCLC cells to seed distant metastases. Accordingly, formation of spontaneous distant metastases was reduced by 50% when OH-1 cells were xenografted into E-/P-selectin-deficient mice compared with wild type mice (p = 0.0181). However, as metastasis formation was not completely abrogated in selectin deficient mice, we concluded that this adhesion cascade is redundant and that other molecules of this cascade mediate metastasis formation as well. Using several of these adhesion molecules as interaction partners presumably make SCLC cells so highly metastatic.     

Lipid profiling of FPLC-separated lipoprotein fractions by electrospray ionization tandem mass spectrometry

Glycerophospholipid and sphingolipid species and their bioactive metabolites are important regulators of lipoprotein and cell function. The aim of the study was to develop a method for lipid species profiling of separated lipoprotein classes. Human serum lipoproteins VLDL, LDL, and HDL of 21 healthy fasting blood donors were separated by fast performance liquid chromatography (FPLC) from 50 microl serum. Subsequently, phosphatidylcholine (PC), lysophosphatidylcholine, sphingomyelin (SM), ceramide (CER), phosphatidylethanolamine (PE), PE-based plasmalogen (PE-pl), cholesterol, and cholesteryl ester (CE) content of the separated lipoproteins was quantified by electrospray ionization tandem mass spectrometry (ESI-MS/MS). Analysis of FPLC fractions with PAGE demonstrated that albumin partially coelutes with HDL fractions. However, analysis of an HDL deficient serum (Tangier disease) showed that only lysophosphatidylcholine, but none of the other lipids analyzed, exhibited a significant coelution with the albumin containing fractions. Approximately 60% of lipoprotein CER were found in LDL fractions and 60% of PC, PE, and plasmalogens in HDL fractions. VLDL, LDL, and HDL displayed characteristic lipid class and species pattern. The developed method provides a detailed lipid class and species composition of lipoprotein fractions and may serve as a valuable tool to identify alterations of lipoprotein lipid species profiles in disease with a reasonable experimental effort.     

Biotransformation and reduction of estrogenicity of bisphenol A by the biphenyl-degrading Cupriavidus basilensis

The biphenyl-degrading Gram-negative bacterium Cupriavidus basilensis (formerly Ralstonia sp.) SBUG 290 uses various aromatic compounds as carbon and energy sources and has a high capacity to transform bisphenol A (BPA), which is a hormonally active substance structurally related to biphenyl. Biphenyl-grown cells initially hydroxylated BPA and converted it to four additional products by using three different transformation pathways: (a) formation of multiple hydroxylated BPA, (b) ring fission, and (c) transamination followed by acetylation or dimerization. Products of the ring fission pathway were non-toxic and all five products exhibited a significantly reduced estrogenic activity compared to BPA. Cell cultivation with phenol and especially in nutrient broth (NB) resulted in a reduced biotransformation rate and lower product quantities, and NB-grown cells did not produce all five products in detectable amounts. Thus, the question arose whether enzymes of the biphenyl degradation pathway are involved in the transformation of BPA and was addressed by proteomic analyses.