Stimulation of platelet apoptosis by peptidoglycan from Staphylococcus aureus 113

Peptidoglycan (PGN), a component of bacterial cell wall and belonging to "Microbe-Associated Molecular Patterns" (MAMP) triggers host reactions contributing to the pathophysiology of infectious disease. Host cell responses to PGN exposure include apoptosis. Bacterial infections may result in activation of blood platelets and thrombocytopenia. The present study explored, whether HPLC-purified fractions of PGNs from Staphylococcus aureus 113 triggers apoptosis of platelets. To this end platelets were exposed to PGN fractions and annexin-V binding determined to depict cell membrane scrambling, DiOC6 fluorescence to estimate depolarization of mitochondrial potential, Fluo-3AM staining for intracellular Ca(2+) activity ([Ca(2+)](i)) and immunofluorescence to quantify protein abundance of active caspase-3. As a result, a 30?min exposure to monomeric fraction (mPGN) (≥50?ng/ml) was followed by annexin-V binding, paralleled by increase of [Ca(2+)](i), mitochondrial depolarization, caspase-3 activation and integrin α(IIb)β(3) upregulation. The annexin-V binding was significantly blunted by anti-TLR-2 antibodies, in absence of extracellular Ca(2+), and by pancaspase inhibitor zVAD-FMK (1?μM). In conclusion, PGN triggers apoptosis of platelets in activation-dependent manner, characterized by mitochondrial depolarization, caspase-3 activation and cell membrane scrambling.     

Lipopolysaccharide-sensitive H+ current in dendritic cells

Dendritic cells (DCs) are the most potent antigen-presenting cells equipped to transport antigens from the periphery to lymphoid tissues and to present them to T cells. Ligation of Toll-like receptor 4 (TLR4), expressed on the DC surface, by lipopolysaccharides (LPS), elements of the Gram-negative bacteria outer wall, induces DC maturation. Initial steps of maturation include stimulation of antigen endocytosis and enhanced reactive oxygen species (ROS) production with eventual downregulation of endocytic capacity in fully matured DCs. ROS production depends on NADPH oxidase (NOX2), the activity of which requires continuous pH and charge compensation. The present study demonstrates, for the first time, the functional expression of voltage-gated proton (Hv1) channels in mouse bone marrow-derived DCs. In whole cell patch-clamp experiments, we recorded Zn(2+) (50 μM)-sensitive outwardly rectifying currents activated upon depolarization, which were highly selective for H(+), with the reversal potential shift of 38 mV per pH unit. The threshold voltage of activation (V(threshold)) was dependent on the pH gradient and was close to the empirically predicted V(threshold) for the Hv1 currents. LPS (1 μg/ml) had bimodal effects on Hv1 channels: acute LPS treatment increased Hv1 channel activity, whereas 24 h of LPS incubation significantly inhibited Hv1 currents and decreased ROS production. Activation of H(+) currents by acute application of LPS was abolished by PKC inhibitor GFX (10 nM). According to electron current measurements, acute LPS application was associated with increased NOX2 activity.     

cNMP-AMs mimic and dissect bacterial nucleotidyl cyclase toxin effects

In addition to the well-known second messengers cAMP and cGMP, mammalian cells contain the cyclic pyrimidine nucleotides cCMP and cUMP. The Pseudomonas aeruginosa toxin ExoY massively increases cGMP and cUMP in cells, whereas the Bordetella pertussis toxin CyaA increases cAMP and, to a lesser extent, cCMP. To mimic and dissect toxin effects, we synthesized cNMP-acetoxymethylesters as prodrugs. cNMP-AMs rapidly and effectively released the corresponding cNMP in cells. The combination of cGMP-AM plus cUMP-AM mimicked cytotoxicity of ExoY. cUMP-AM and cGMP-AM differentially activated gene expression. Certain cCMP and cUMP effects were independent of the known cNMP effectors protein kinases A and G and guanine nucleotide exchange factor Epac. In conclusion, cNMP-AMs are useful tools to mimic and dissect bacterial nucleotidyl cyclase toxin effects.     

In Vitro and In Vivo Neuronal Electrotaxis: A Potential Mechanism for Restoration?

Electrical brain stimulation used to treat a variety of neurological and psychiatric diseases is entering a new period. The technique is well established and the potential complications are well known and generally manageable. Recent studies demonstrated that electrical fields (EFs) can enhance neuroplasticity-related processes. EFs applied in the physiological range induce migration of different neural cell types from different species in vitro. There are some evidences that also the speed and directedness of cell migration are enhanced by EFs. However, it is still unclear how electrical signals from the extracellular space are translated into intracellular actions resulting in the so-called electrotaxis phenomenon. Here, we aim to provide a comprehensive review of the data on responses of cells to electrical stimulation and the relation to functional recovery.     

Sphingosine 1-phosphate is a ligand of the human gpr3, gpr6 and gpr12 family of constitutively active G protein-coupled receptors

Five G protein-coupled receptors (GPCRs) for the lysophospholipid sphingosine 1-phosphate (S1P) have been cloned and characterized so far. We report here about the identification of gpr3, gpr6 and gpr12 as additional members of the S1P-GPCR family. When expressed transiently in HEK293 cells, gpr3, gpr6 and gpr12 confer constitutive activation of adenylate cyclase (AC) similar in amplitude to that seen with fully activated G(alpha)(s)-coupled receptors. Culturing the transfected cells in medium with charcoal-stripped serum (devoid of lipids) significantly reduces cyclic adenosine monophosphate (cAMP) levels, suggesting a lipid-like ligand. A library containing 200 bioactive lipids was applied in functional assays recording intracellular Ca(2+) mobilization. S1P and dihydrosphingosine 1-phosphate (DHS1P) were identified as functional activators exhibiting nanomolar EC(50) values. In the presence of the S1P and LPA receptor antagonist suramin, gpr3-, gpr6- and gpr12-mediated intracellular Ca(2+) mobilization via S1P is enhanced. Besides constitutive activation of G(alpha)(s) type of G proteins, all three receptors are capable of constitutively activating inhibitory G(alpha)(i/o) proteins: (i) in the presence of pertussis toxin, gpr3-, gpr6- and gpr12-mediated stimulation of AC is enhanced; and (ii) overexpression of G(alpha)(i) significantly reduces the stimulatory action on intracellular cAMP levels. Agonist (S1P)-mediated internalization can be visualized in intact HEK293 cells using a gpr6 green fluorescent protein (GFP) fusion protein. In summary, our data suggest that gpr3, gpr6 and gpr12 are a family of constitutively active receptors with dual coupling to G(alpha)(s) and G(alpha)(i) type of G proteins. Constitutive activation of AC and mobilization of [Ca(2+)](i) can be modulated by the sphingophospholipids S1P and DHS1P, adding three additional members to the family of S1P receptors.     

In vitro myometrial inhibition by the partitioned aqueous fraction of Anthocleista djalonensis leaves

This study investigated the effect on the uterus of the aqueous fraction of the partitioned methanol crude extract of the leaves of Anthocleista djalonensis (AD) and the possible mechanism of AD activity. AD inhibited the concentration-response curves induced by oxytocin and CaCl2 on the rat uterus in vitro and significantly reduced the EC50 in a concentration-dependent manner (p?< 0.05). A similar effect was observed with salbutamol and verapamil on the concentration-response curves obtained for oxytocin and CaCl2. The inhibitory effect of AD was not attenuated in the presence of propranolol. AD, salbutamol, and verapamil also produced a concentration-dependent relaxation on K+-induced sustained uterine contraction. In Ca2+-free medium, AD and salbutamol similarly inhibited oxytocin-induced contraction, but verapamil failed to produce this effect. The present results suggest that AD, being a mixture of phytochemicals, probably exerts inhibitory activity on in vitro uterine contractions of the nonpregnant, diethylstilboestrol-treated rat by multiple mechanisms that do not involve interaction with β-adrenergic receptors and do not solely depend on inhibition of calcium influx.     

Glycopeptide biosynthesis in the context of basic cellular functions

Using molecular genetics, biochemistry and organic chemistry the biosynthesis of glycopeptides has been elucidated in detail. It can be categorised in three parts: precursor supply, linking of the peptide backbone and modification reactions. The important steps of the biosynthesis are carried out at a multi-enzyme complex consisting of three non-ribosomal peptide synthetases (NRPS), three oxygenases and one halogenase. Novel derivatives can be generated by precursor-directed biosynthesis or combinatorial approaches and the knowledge can be used to optimise the yield of production by metabolic engineering approaches. To protect themselves glycopeptide producers seem to have developed strategies which may differ from those of the resistant pathogens.     

Synthetic biology of secondary metabolite biosynthesis in actinomycetes: Engineering precursor supply as a way to optimize antibiotic production

Actinomycetes are a rich source for the synthesis of medically and technically useful natural products. The genes encoding the enzymes for their biosynthesis are normally organized in gene clusters, which include also the information for resistance (in the case of antibacterial compounds), regulation, and transport. This facilitates the manipulation of such pathways by molecular genetic techniques. Recent advances in DNA sequencing and analytical chemistry revealed that not only new strains isolated from yet unexplored habitats, but also already known strains possess a large potential for the synthesis of novel compounds. Synthetic Biology now offers a new perspective to exploit this potential further by generating novel pathways, and thereby novel products, by combining different biosynthetic steps originating from different bacteria. The supply of precursors, which are subsequently incorporated into the final product, is often already organized in a modular manner in nature and may directly be exploited for Synthetic Biology. Here we report examples for the synthesis of building blocks and possibilities to modify and optimize antibiotic biosynthesis, exemplary for the synthesis of the manipulation of the synthesis of the glycopeptide antibiotic balhimycin.     

Conditional inactivation of nicastrin restricts amyloid deposition in an Alzheimer's disease mouse model

Production of Aβ by γ‐secretase is a key event in Alzheimer's disease (AD). The γ‐secretase complex consists of presenilin (PS) 1 or 2, nicastrin (ncstn), Pen‐2, and Aph‐1 and cleaves type I transmembrane proteins, including the amyloid precursor protein (APP). Although ncstn is widely accepted as an essential component of the complex required for γ‐secretase activity, recent in vitro studies have suggested that ncstn is dispensable for APP processing and Aβ production. The focus of this study was to answer this controversy and evaluate the role of ncstn in Aβ generation and the development of the amyloid‐related phenotype in the mouse brain. To eliminate ncstn expression in the mouse brain, we used a ncstn conditional knockout mouse that we mated with an established AD transgenic mouse model (5XFAD) and a neuronal Cre‐expressing transgenic mouse (CamKIIα‐iCre), to generate AD mice (5XFAD/CamKIIα‐iCre/ncstn^f/f mice) where ncstn was conditionally inactivated in the brain. 5XFAD/CamKIIα‐iCre/ncstn^f/f mice at 10 week of age developed a neurodegenerative phenotype with a significant reduction in Aβ production and formation of Aβ aggregates and the absence of amyloid plaques. Inactivation of nctsn resulted in substantial accumulation of APP‐CTFs and altered PS1 expression. These results reveal a key role for ncstn in modulating Aβ production and amyloid plaque formation in vivo and suggest ncstn as a target in AD therapeutics.     

Quality Evaluation of Methyl Binding Domain Based Kits for Enrichment DNA-Methylation Sequencing

DNA-methylation is an important epigenetic feature in health and disease. Methylated sequence capturing by Methyl Binding Domain (MBD) based enrichment followed by second-generation sequencing provides the best combination of sensitivity and cost-efficiency for genome-wide DNA-methylation profiling. However, existing implementations are numerous, and quality control and optimization require expensive external validation. Therefore, this study has two aims: 1) to identify a best performing kit for MBD-based enrichment using independent validation data, and 2) to evaluate whether quality evaluation can also be performed solely based on the characteristics of the generated sequences. Five commercially available kits for MBD enrichment were combined with Illumina GAIIx sequencing for three cell lines (HCT15, DU145, PC3). Reduced representation bisulfite sequencing data (all three cell lines) and publicly available Illumina Infinium BeadChip data (DU145 and PC3) were used for benchmarking. Consistent large-scale differences in yield, sensitivity and specificity between the different kits could be identified, with Diagenode''s MethylCap kit as overall best performing kit under the tested conditions. This kit could also be identified with the Fragment CpG-plot, which summarizes the CpG content of the captured fragments, implying that the latter can be used as a tool to monitor data quality. In conclusion, there are major quality differences between kits for MBD-based capturing of methylated DNA, with the MethylCap kit performing best under the used settings. The Fragment CpG-plot is able to monitor data quality based on inherent sequence data characteristics, and is therefore a cost-efficient tool for experimental optimization, but also to monitor quality throughout routine applications.