Reduction of a tetrazolium salt, CTC, by intact HepG2 human hepatoma cells: subcellular localisation of reducing systems

AbstractCell-mediated reduction of tetrazolium salts, including MTT, XTT, MTS, NBT, NTV, INT, in the presence or absence of intermediate electron carriers is used as a convenient test for animal or bacterial cell viability. Bioreduction of tetrazolium is considered an alternative to a clonogenic assay and a thymidine incorporation assay. However, correlation between clonogenic potential and capacity to reduce tetrazolium has not been demonstrated convincingly. Moreover, despite a wide use of tetrazolium viability assays, the mechanism and subcellular localisation of reducing systems or species in viable intact cells have not been fully elucidated. We report evidence indicating that a tetrazolium salt CTC can be reduced in the presence as well as in the absence of an electron carrier by viable HepG2 human hepatoma cells. CTC-formazan is formed within or at the outer surface of plasma membranes. We hypothesise that in the presence of an electron carrier the electron donors active in the reduction of CTC are located in the intracellular compartment, as well as in plasma membranes. However, in the absence of an electron carrier, the reduction occurs primarily via a plasma membrane-associated enzymatic system or species

Low Level Phosphorylation of Histone H2AX on Serine 139 (gammaH2AX) Is Not Associated with DNA Double-Strand Breaks

Phosphorylation of histone H2AX on serine 139 (γH2AX) is an early step in cellular response to a DNA double-strand break (DSB). γH2AX foci are generally regarded as markers of DSBs. A growing body of evidence demonstrates, however, that while induction of DSBs always brings about phosphorylation of histone H2AX, the reverse is not true - the presence of γH2AX foci should not be considered an unequivocal marker of DNA double-strand breaks. We studied DNA damage induced in A549 human lung adenocarcinoma cells by topoisomerase type I and II inhibitors (0.2 μM camptothecin, 10 μM etoposide or 0.2 μM mitoxantrone for 1 h), and using 3D high resolution quantitative confocal microscopy, assessed the number, size and the integrated intensity of immunofluorescence signals of individual γH2AX foci induced by these drugs. Also, investigated was spatial association between γH2AX foci and foci of 53BP1, the protein involved in DSB repair, both in relation to DNA replication sites (factories) as revealed by labeling nascent DNA with EdU. Extensive 3D and correlation data analysis demonstrated that γH2AX foci exhibit a wide range of sizes and levels of H2AX phosphorylation, and correlate differently with 53BP1 and DNA replication. This is the first report showing lack of a link between low level phosphorylation γH2AX sites and double-strand DNA breaks in cells exposed to topoisomerase I or II inhibitors. The data are discussed in terms of mechanisms that may be involved in formation of γH2AX sites of different sizes and intensities

SEM-EDS and X-ray micro computed tomography studies of skeletal surface pattern and body structure in the freshwater sponge Spongilla lacustris collected from Goczalkowice reservoir habit (Southern Poland)

Introduction. Freshwater sponges are common animals of most aquatic ecosystems. They feed by filtering small particles from the water, and so are thought to be sensitive indicators of pollution. Sponges are strongly associated with the abiotic environment and are therefore used as bioindicators for monitoring of water quality in water habitats. Among the freshwater sponges, Spongilla lacustris is one of the classic models used to study evolution, gene regulation, development, physiology and structural biology in animal water systems. It is also important in diagnostic of aquatic environments. The aim of this study was to characterize and visualize three-dimensional architecture of sponge body and measure skeleton elements of S. lacustris from Goczalkowice reservoir for identification purposes. Material and methods. The scanning electron microscopy with an energy dispersive X-ray microanalysis (SEM- -EDS) and X-ray micro computed tomography (micro-CT) were used to provide non-invasive visualization of the three-dimensional architecture of Spongilla lacustris body. Results. We showed that sponge skeleton was not homogeneous in composition and comprised several forms of skeleton organization. Ectosomal skeleton occurred as spicular brushes at apices of primary fibres with cementing spongin material. Choanosomal skeletal architecture was alveolate with pauci- to multispicular primary fibres connected by paucispicular transverse fibres, made by megascleres embedded in a scanty spongin matrix both in the choanosome and at the sponge surface. In contrast, microscleres were irregularly scattered in choanosome and skeletal surface. Furthermore, SEM-EDS studies showed that the distribution of silica in megascleres and microscleres was observed along the spicules and sponge surface areas. Conclusions. In conclusion, we showed that the combination of SEM-EDS and micro-CT microscopy techniques allowed obtaining a complete picture of the sponge spatial architecture

Fibroblast growth factor homologous factor 1 interacts with NEMO to regulate NF-κB signaling in neurons.

Neuronal survival and plasticity critically depend on constitutive activity of the transcription factor nuclear factor-κB (NF-κB). We here describe a role for a small intracellular fibroblast growth factor homologue, the fibroblast growth factor homologous factor 1 (FHF1/FGF12), in the regulation of NF-κB activity in mature neurons. FHFs have previously been described to control neuronal excitability, and mutations in FHF isoforms give rise to a form of progressive spinocerebellar ataxia. Using a protein-array approach, we identified FHF1b as a novel interactor of the canonical NF-κB modulator IKKγ/NEMO. Co-immunoprecipitation, pull-down and GAL4-reporter experiments, as well as proximity ligation assays, confirmed the interaction of FHF1 and NEMO and demonstrated that a major site of interaction occurred within the axon initial segment. Fhf1 gene silencing strongly activated neuronal NF-κB activity and increased neurite lengths, branching patterns and spine counts in mature cortical neurons. The effects of FHF1 on neuronal NF-κB activity and morphology required the presence of NEMO. Our results imply that FHF1 negatively regulates the constitutive NF-κB activity in neurons

Latrepirdine is a potent activator of AMP-activated protein kinase and reduces neuronal excitability.

Latrepirdine/Dimebon is a small-molecule compound with attributed neurocognitive-enhancing activities, which has recently been tested in clinical trials for the treatment of Alzheimer\u27s and Huntington\u27s disease. Latrepirdine has been suggested to be a neuroprotective agent that increases mitochondrial function, however the molecular mechanisms underlying these activities have remained elusive. We here demonstrate that latrepirdine, at (sub)nanomolar concentrations (0.1 nM), activates the energy sensor AMP-activated protein kinase (AMPK). Treatment of primary neurons with latrepirdine increased intracellular ATP levels and glucose transporter 3 translocation to the plasma membrane. Latrepirdine also increased mitochondrial uptake of the voltage-sensitive probe TMRM. Gene silencing of AMPKα or its upstream kinases, LKB1 and CaMKKβ, inhibited this effect. However, studies using the plasma membrane potential indicator DisBAC2(3) demonstrated that the effects of latrepirdine on TMRM uptake were largely mediated by plasma membrane hyperpolarization, precluding a purely \u27mitochondrial\u27 mechanism of action. In line with a stabilizing effect of latrepirdine on plasma membrane potential, pretreatment with latrepirdine reduced spontaneous Ca(2+) oscillations as well as glutamate-induced Ca(2+) increases in primary neurons, and protected neurons against glutamate toxicity. In conclusion, our experiments demonstrate that latrepirdine is a potent activator of AMPK, and suggest that one of the main pharmacological activities of latrepirdine is a reduction in neuronal excitability

Sphingosine kinases regulate ER contacts with late endocytic organelles and cholesterol trafficking

Membrane contact sites (MCS), close membrane apposition between organelles, are platforms for interorganellar transfer of lipids including cholesterol, regulation of lipid homeostasis, and co-ordination of endocytic trafficking. Sphingosine kinases (SphKs), two isoenzymes that phosphorylate sphingosine to the bioactive sphingosine-1-phosphate (S1P), have been implicated in endocytic trafficking. However, the physiological functions of SphKs in regulation of membrane dynamics, lipid trafficking and MCS are not known. Here, we report that deletion of SphKs decreased S1P with concomitant increases in its precursors sphingosine and ceramide, and markedly reduced endoplasmic reticulum (ER) contacts with late endocytic organelles. Expression of enzymatically active SphK1, but not catalytically inactive, rescued the deficit of these MCS. Although free cholesterol accumulated in late endocytic organelles in SphK null cells, surprisingly however, cholesterol transport to the ER was not reduced. Importantly, deletion of SphKs promoted recruitment of the ER-resident cholesterol transfer protein Aster-B (also called GRAMD1B) to the plasma membrane (PM), consistent with higher accessible cholesterol and ceramide at the PM, to facilitate cholesterol transfer from the PM to the ER. In addition, ceramide enhanced in vitro binding of the Aster-B GRAM domain to phosphatidylserine and cholesterol liposomes. Our study revealed a previously unknown role for SphKs and sphingolipid metabolites in governing diverse MCS between the ER network and late endocytic organelles versus the PM to control the movement of cholesterol between distinct cell membranes

Activation-induced chromatin reorganization in neurons depends on HDAC1 activity

Spatial chromatin organization is crucial for transcriptional regulation and might be particularly important in neurons since they dramatically change their transcriptome in response to external stimuli. We show that stimulation of neurons causes condensation of large chromatin domains. This phenomenon can be observed in vitro in cultured rat hippocampal neurons as well as in vivo in the amygdala and hippocampal neurons. Activity-induced chromatin condensation is an active, rapid, energy-dependent, and reversible process. It involves calcium-dependent pathways but is independent of active transcription. It is accompanied by the redistribution of posttranslational histone modifications and rearrangements in the spatial organization of chromosome territories. Moreover, it leads to the reorganization of nuclear speckles and active domains located in their proximity. Finally, we find that the histone deacetylase HDAC1 is the key regulator of this process. Our results suggest that HDAC1-dependent chromatin reorganization constitutes an important level of transcriptional regulation in neurons.publishedVersio

Activation-induced chromatin reorganization in neurons depends on HDAC1 activity

Spatial chromatin organization is crucial for transcriptional regulation and might be particularly important in neurons since they dramatically change their transcriptome in response to external stimuli. We show that stimulation of neurons causes condensation of large chromatin domains. This phenomenon can be observed in vitro in cultured rat hippocampal neurons as well as in vivo in the amygdala and hippocampal neurons. Activity-induced chromatin condensation is an active, rapid, energy-dependent, and reversible process. It involves calcium-dependent pathways but is independent of active transcription. It is accompanied by the redistribution of posttranslational histone modifications and rearrangements in the spatial organization of chromosome territories. Moreover, it leads to the reorganization of nuclear speckles and active domains located in their proximity. Finally, we find that the histone deacetylase HDAC1 is the key regulator of this process. Our results suggest that HDAC1-dependent chromatin reorganization constitutes an important level of transcriptional regulation in neurons.This work was supported by the National Science Centre grant nos. UMO-2015/18/E/NZ3/00730 (A.M., A.G., H.S.N., E.J. and Y.Y.), 2014/15/N/NZ2/00379 and 2017/24/T/NZ2/00307 (P.T.), 2019/35/O/ST6/02484 (D.P. and G.B.), and 2014/14/M/NZ4/00561 (K.H.O. and R.K.F.). B.W. and B.G. were supported by the Foundation for Polish Science TEAM-TECH Core Facility project “NGS platform for comprehensive diagnostics and personalized therapy in neuro-oncology,” Foundation for Polish Science co-financed by the European Union under the European Regional Development Fund (TEAM to D.P.). A.M.G. was supported by the H2020-MSCA-COFUND-2014 grant Bio4Med (GA no. 665735).Peer reviewe