Inhibition of histone deacetylation protects wild-type but not gelsolin-deficient neurons from oxygen/glucose deprivation

Histone acetylation and deacetylation participate in the epigenetic regulation of gene expression. In this paper, we demonstrate that pre-treatment with the histone deacetylation inhibitor trichostatin A (TSA) enhances histone acetylation in primary cortical neurons and protects against oxygen/glucose deprivation, a model for ischaemic cell death in vitro. The actin-binding protein gelsolin was identified as a mediator of neuroprotection by TSA. TSA enhanced histone acetylation of the gelsolin promoter region, and up-regulated gelsolin messenger RNA and protein expression in a dose- and time-dependent manner. Double-label confocal immunocytochemistry visualized the up-regulation of gelsolin and histone acetylation within the same neuron. Together with gelsolin up-regulation, TSA pre-treatment decreased levels of filamentous actin. The neuroprotective effect of TSA was completely abolished in neurons lacking gelsolin gene expression. In conclusion, we demonstrate that the enhancement of gelsolin gene expression correlates with neuroprotection induced by the inhibition of histone deacetylation.     

ISCOMATRIX adjuvant combines immune activation with antigen delivery to dendritic cells in vivo leading to effective cross-priming of CD8+ T cells

Cancer vaccines aim to induce CTL responses against tumors. Challenges for vaccine design are targeting Ag to dendritic cells (DCs) in vivo, facilitating cross-presentation, and conditioning the microenvironment for Th1 type immune responses. In this study, we report that ISCOM vaccines, which consist of ISCOMATRIX adjuvant and protein Ag, meet these challenges. Subcutaneous injection of an ISCOM vaccine in mice led to a substantial influx and activation of innate and adaptive immune effector cells in vaccine site-draining lymph nodes (VDLNs) as well as IFN-γ production by NK and NKT cells. Moreover, an ISCOM vaccine containing the model Ag OVA (OVA/ISCOM vaccine) was efficiently taken up by CD8α(+) DCs in VDLNs and induced their maturation and IL-12 production. Adoptive transfer of transgenic OT-I T cells revealed highly efficient cross-presentation of the OVA/ISCOM vaccine in vivo, whereas cross-presentation of soluble OVA was poor even at a 100-fold higher concentration. Cross-presenting activity was restricted to CD8α(+) DCs in VDLNs, whereas Langerin(+) DCs and CD8α(-) DCs were dispensable. Remarkably, compared with other adjuvant systems, the OVA/ISCOM vaccine induced a high frequency of OVA-specific CTLs capable of tumor cell killing in different tumor models. Thus, ISCOM vaccines combine potent immune activation with Ag delivery to CD8α(+) DCs in vivo for efficient induction of CTL responses.     

Staphylococcus aureus CidA and LrgA proteins exhibit holin-like properties

The Staphylococcus aureus cid and lrg operons are known to be involved in biofilm formation by controlling cell lysis and the release of genomic DNA, which ultimately becomes a structural component of the biofilm matrix. Although the molecular mechanisms controlling cell death and lysis are unknown, it has been hypothesized that the cidA and lrgA genes encode holin- and antiholin-like proteins and function to regulate these processes similarly to bacteriophage-induced death and lysis. In this study, we focused on the biochemical and molecular characterization of CidA and LrgA with the goal of testing the holin model. First, membrane fractionation and fluorescent protein fusion studies revealed that CidA and LrgA are membrane-associated proteins. Furthermore, similarly to holins, CidA and LrgA were found to oligomerize into high-molecular-mass complexes whose formation was dependent on disulfide bonds formed between cysteine residues. To determine the function of disulfide bond-dependent oligomerization of CidA, an S. aureus mutant in which the wild-type copy of the cidA gene was replaced with the cysteine mutant allele was generated. As determined by β-galactosidase release assays, this mutant exhibited increased cell lysis during stationary phase, suggesting that oligomerization has a negative impact on this process. When analyzed for biofilm development and maturation, this mutant displayed increased biofilm adhesion in a static assay and a greater amount of dead-cell accumulation during biofilm maturation. These studies support the model that CidA and LrgA proteins are bacterial holin-/antiholin-like proteins that function to control cell death and lysis during biofilm development.     

Predicting Chemical Environments of Bacteria from Receptor Signaling

Sensory systems have evolved to respond to input stimuli of certain statistical properties, and to reliably transmit this information through biochemical pathways. Hence, for an experimentally well-characterized sensory system, one ought to be able to extract valuable information about the statistics of the stimuli. Based on dose-response curves from in vivo fluorescence resonance energy transfer (FRET) experiments of the bacterial chemotaxis sensory system, we predict the chemical gradients chemotactic Escherichia coli cells typically encounter in their natural environment. To predict average gradients cells experience, we revaluate the phenomenological Weber''s law and its generalizations to the Weber-Fechner law and fold-change detection. To obtain full distributions of gradients we use information theory and simulations, considering limitations of information transmission from both cell-external and internal noise. We identify broad distributions of exponential gradients, which lead to log-normal stimuli and maximal drift velocity. Our results thus provide a first step towards deciphering the chemical nature of complex, experimentally inaccessible cellular microenvironments, such as the human intestine.     

Influence of Pigment Epithelium-Derived Factor on Outcome after Striatal Cerebral Ischemia in the Mouse

We here suggest that pigment epithelium-derived factor (PEDF) does not have an effect on lesion size, behavioral outcome, cell proliferation, or cell death after striatal ischemia in the mouse. PEDF is a neurotrophic factor with neuroprotective, antiangiogenic, and antipermeability effects. It influences self-renewal of neural stem cells and proliferation of microglia. We investigated whether intraventricular infusion of PEDF reduces infarct size and cell death, ameliorates behavioral outcome, and influences cell proliferation in the one-hour middle cerebral artery occlusion (MCAO) mouse model of focal cerebral ischemia. C57Bl6/N mice were implanted with PEDF or artificial cerebrospinal fluid (control) osmotic pumps and subjected to 60-minute MCAO 48 hours after pump implantation. They received daily BrdU injections for 7 days after MCAO in order to investigate cell proliferation. Infarct volumes were determined 24 hours after reperfusion using magnetic resonance imaging. We removed the pumps on day 5 and performed behavioral testing between day 7 and 21. Immunohistochemical staining was performed to determine the effect of PEDF on cell proliferation and cell death. Our model produced an ischemic injury confined solely to striatal damage. We detected no reduction in infarct sizes and cell death in PEDF- vs. CSF-infused MCAO mice. Behavioral outcome and cell proliferation did not differ between the groups. However, we cannot exclude that PEDF might work under different conditions in stroke. Further studies will elucidate the effect of PEDF treatment on cell proliferation and behavioral outcome in moderate to severe ischemic injury in the brain.     

Oligodeoxynucleotides enhance lipopolysaccharide-stimulated synthesis of tumor necrosis factor: dependence on phosphorothioate modification and reversal by heparin.

BACKGROUND: Specific inhibition of target proteins by antisense oligodeoxynucleotides is an extensively studied experimental approach. This technique is currently being tested in clinical trials applying phosphorothioate-modified oligonucleotides as therapeutic agents. These polyanionic molecules, however, may also exert non-antisense-mediated effects. MATERIALS AND METHODS: We examined the influence of oligonucleotides on lipopolysaccharide (LPS)-stimulated tumor necrosis factor alpha (TNF alpha) synthesis in freshly isolated human peripheral blood mononuclear cells. Oligonucleotides (18 mer) with different degrees of phosphorothioate modification were studied. RESULTS: The addition of phosphorothioate oligonucleotides (5 microM) caused amplification of TNF synthesis of up to 410% compared with the control with LPS alone. Without LPS stimulation, phosphorothioate oligonucleotides did not induce TNF production. We demonstrate that the enhancement of LPS-stimulated TNF production by phosphorothioate oligonucleotides does not rely on the intracellular presence of oligonucleotides and is not mediated by LPS contamination. Partially phosphorothioate-modified oligonucleotides and unmodified oligonucleotides did not increase TNF synthesis. High concentrations of the polyanion heparin reversed the oligonucleotide-induced enhancement of TNF synthesis. CONCLUSIONS: The data suggest that amplification of TNF synthesis may be caused by binding of the polyanionic phosphorothioate oligonucleotide to cationic sites on the cell surface. Such binding sites have been proposed for polyanionic glycoaminoglycans of the extracellular matrix, which have also been described to augment LPS-stimulated TNF synthesis. The present results are relevant to all in vitro studies attempting to influence protein synthesis in monocytes by using phosphorothioate oligonucleotides. The significance of our findings for in vivo applications of phosphorothioates in situations where there is a stimulus for TNF synthesis, such as in sepsis, should be elucidated.     

Ammonium and urea as nitrogen sources for bromeliads

Epiphytic bromeliads have no contact with the pedosphere, so they need to draw their nutrients from the atmosphere as well as from the host tree and animal debris. Terrestrial bromeliads, like Ananas comosus, generally depend on the soil as their main nutrient source. The aim of this study was to investigate and compare some aspects of the nitrogen metabolism of two bromeliads with different growth habits: Ananas comosus, a terrestrial bromeliad, and Vriesea gigantea, an epiphytic tank bromeliad. Nitrogen-starved plants were grown in vitro for 3, 7, 15, 30, and 60 days, either with 5 mmol L⁻¹ ammonium [(NH₄)₂SO₄] or urea as the sole nitrogen source. When NH₄⁺ was supplied to the plants, it stimulated a faster increase of chlorophyll content in A. comosus than in V. gigantea. In the presence of urea, after 15 days of the plants in culture, there was a significant increase in tissue free-NH₄⁺ and total amino acids for V. gigantea only. V. gigantea presented a higher level of total free amino acids than A. comosus when nitrogen was supplied to the plants. Asparagine was the main amino acid accumulated in both bromeliads when plants were transferred to the medium with nitrogen. When the ratio of the main individual free amino acids between the bromeliads grown in NH₄⁺ and urea was compared, values such as 7.2 for asparagine, 5.3 for glutamate, and 1.8 for aspartate in A. comosus, and values such as 2.3 for asparagine, 1.1 for glutamate and 0.7 for aspartate in V. gigantea were observed, demonstrating that the last is more efficient in assimilating urea. The results prompted us to support the idea that V. gigantea, an epiphytic tank bromeliad, is better adapted to absorb and assimilate organic nitrogen, such as urea, while A. comosus, a terrestrial plant, is better adapted to inorganic nitrogen forms, such as ammonium. The natural exposure of tank bromeliads to urea is discussed in the paper.     

N-aryl-piperidine-4-carboxamides as a novel class of potent inhibitors of MALT1 proteolytic activity

Starting from a weak screening hit, potent and selective inhibitors of the MALT1 protease function were elaborated. Advanced compounds displayed high potency in biochemical and cellular assays. Compounds showed activity in a mechanistic Jurkat T cell activation assay as well as in the B-cell lymphoma line OCI-Ly3, which suggests potential use of MALT1 inhibitors in the treatment of autoimmune diseases as well as B-cell lymphomas with a dysregulated NF-κB pathway. Initially, rat pharmacokinetic properties of this compound series were dominated by very high clearance which could be linked to amide cleavage. Using a rat hepatocyte assay a good in vitro-in vivo correlation could be established which led to the identification of compounds with improved PK properties.