Imaging synaptic inhibition in transgenic mice expressing the chloride indicator, Clomeleon

We describe here a molecular genetic approach for imaging synaptic inhibition. The thy-1 promoter was used to express high levels of Clomeleon, a ratiometric fluorescent indicator for chloride ions, in discrete populations of neurons in the brains of transgenic mice. Clomeleon was functional after chronic expression and provided non-invasive readouts of intracellular chloride concentration ([Cl(-)](i)) in brain slices, allowing us to quantify age-dependent declines in resting [Cl(-)](i) during neuronal development. Activation of hippocampal interneurons caused [Cl(-)](i) to rise transiently in individual postsynaptic pyramidal neurons. [Cl(-)](i) increased in direct proportion to the amount of inhibitory transmission, with peak changes as large as 4 mM. Integrating responses over populations of pyramidal neurons allowed sensitive detection of synaptic inhibition. Thus, Clomeleon imaging permits non-invasive, spatiotemporally resolved recordings of [Cl(-)](i) in a large variety of neurons, opening up new opportunities for imaging synaptic inhibition and other forms of chloride signaling.     

A Novel TRAF6 binding site in MALT1 defines distinct mechanisms of NF-kappaB activation by API2middle dotMALT1 fusions

The recurrent translocation t(11;18)(q21;q21) associated with mucosa-associated lymphoid tissue (MALT) lymphoma results in the expression of an API2.MALT1 fusion protein that constitutively activates NF-kappaB. The first baculovirus IAP repeat (BIR) domain of API2 and the C terminus of MALT1, which contains its caspase-like domain, are present in all reported fusion variants and interact with TRAF2 and TRAF6, respectively, suggesting their contribution to NF-kappaB signaling by API2.MALT1. Also, the involvement of BCL10 has been suggested via binding to BIR1 of API2 and via its interaction with the immunoglobulin domains of MALT1, present in half of the fusion variants. However, conflicting reports exist concerning their roles in API2.MALT1-induced NF-kappaB signaling. In this report, streptavidin pulldowns of biotinylated API2.MALT1 fusion variants showed that none of the fusion variants interacted with endogenous BCL10; its role in NF-kappaB signaling by API2.MALT1 was further questioned by RNA interference experiments. In contrast, TRAF6 was essential for NF-kappaB activation by all fusion variants, and we identified a novel TRAF6 binding site in the second immunoglobulin domain of MALT1, which enhanced NF-kappaB activation when present in the fusion protein. Furthermore, inclusion of both immunoglobulin domains in API2.MALT1 further enhanced NF-kappaB signaling via intramolecular TRAF6 activation. Finally, binding of TRAF2 to BIR1 contributed to NF-kappaB activation by API2.MALT1, although additional mechanisms involving BIR1-mediated raft association are also important. Taken together, these data reveal distinct mechanisms of NF-kappaB activation by the different API2.MALT1 fusion variants with an essential role for TRAF6.     

Amino-caprolactam derivatives as gamma-secretase inhibitors

A series of amino-caprolactam sulfonamides were developed from a screening hit. Compounds with good in vitro and in vivo gamma-secretase activity are reported.     

Investigation of the bioactivity and biocompatibility of different glass interfaces with hydroxyapatite, fluorohydroxyapatite and 58S bioactive glass

The current review investigates the bioactivity of different glass interfaces created on thin glass cover slips as substrates. The interfaces studied are plain glass, functionalized glass using 0.5 M and 5 M of sodium hydroxide (NaOH) for 24 hrs, and glass coated with bioactive 58S Bioglass (58S). A biomimetic method, involving the exposure of the three interfaces to 1.5 times simulated body fluid (SBF) tests the bioactivity of the interfaces via creation of layer of Hydroxyapatite (HA). Fluorinated SBF will precipitate fluorine doped HA (FHA) on a bioactive interface. Higher concentration of 1.5 times of SBF used in this study intended to accelerate the formation of HA and FHA layer over the substrate. HA and FHA is found to be precipitated on the thinly coated 58S. This paper, study also the thin film coatings of three forms of bioceramics - bioactive 58S, HA and FHA. The study, also proposes to draw a relation between the morphology of HA particles with duration of exposure to SBF, the effects of fluorine on the morphology and the cell interaction with bioactive 58S, HA and FHA interfaces using pre-differentiated osteoblastic MC3T3 cells. The analysis of cells in this study is confined to three parameters that include the attachment, proliferation and viability of cells. Tests employed for the analysis of the thin film coating of HA and FHA is restricted to qualitative X-Ray Diffraction and quantitative Field Emission Scanning Electron Microscope. Other mechanical tests such as shear test are not used to test the mechanical properties of this thin layer, due to the fact that the thin film is too thin for such analysis.     

Mechanism of hydrogen peroxide-induced keratinocyte migration in a scratch-wound model

Recent studies have shown that low concentrations of H₂O₂ are produced endogenously by nonphagocytes after wounding. We observed that H₂O₂ at such concentrations can stimulate proliferation as well as migration of keratinocytes in a scratch-wound assay. Both wounding and H₂O₂ can induce phosphorylation of ERK1/2 via EGFR, but the activation of ERK1/2 by H₂O₂ is more sustained and can last more than 8 h. Sustained ERK1/2 activation is required for the increased proliferation and migration induced by H₂O₂. The p38 MAPK was also found to be phosphorylated upon treatment with H₂O₂ but it was not required for H₂O₂-induced migration or proliferation. Furthermore, it was observed that there is a cross talk between the ERK1/2 and the p38 pathways whereby inhibition of either pathway can lead to activation of the other. As a result, the motogenic effects of H₂O₂ were further enhanced when p38 was inhibited. Our data are consistent with the view that H₂O₂ may play an important signaling role in wound healing.     

Cellular iron depletion weakens induction of heme oxygenase-1 by cadmium

Heme oxygenase-1 is an inducible cytoprotective gene, although its induction by environmental factors is not completely understood. This study aimed to ascertain if specific nutritive factors or related compounds influence heme oxygenase-1 expression. In HCT-116 cells, cadmium increased heme oxygenase-1 enzymatic activity. This effect of cadmium was weaker in cells made iron-deficient with the iron chelator, desferrioxamine, which was associated with repression of heme oxygenase-1 protein and mRNA expression. The repression by desferrioxamine of cadmium-induced heme oxygenase-1 upregulation was reversed upon iron replenishment of the cells. Additionally, it was found that thiol antioxidants inhibited the heme oxygenase-1 upregulation caused by cadmium and also by ethacrynic acid, which each decreased intracellular glutathione as did buthionine sulfoxamine. Interestingly, cadmium and ethacrynic acid increased nuclear translocation of Nrf2 and subsequent heme oxygenase-1 expression, but buthionine sulfoxamine did not. Furthermore, NADPH oxidase inhibitors (diphenyleneiodonium and apocynin, and a superoxide scavenger (Tiron) inhibited cadmium-induced upregulation of heme oxygenase-1. Diphenyleneiodonium was the most potent and inhibited NADPH-cytochrome P450 reductase as well, whereas apocynin and Tiron did not. It is concluded that adequate amounts of iron, which at the atomic level can serve as the pivotal element of heme in NADPH oxidase, must be present in cells to permit what appears to be thiol redox-sensitive, NADPH oxidase-dependent upregulation of heme oxygenase-1. Thus, these findings are significant because they suggest that cells without adequate iron would be unable to fully express the stress gene, heme oxygenase-1, when confronted with the toxic metal, cadmium.     

Engineering microbes to sense and eradicate Pseudomonas aeruginosa,a human pathogen

Synthetic biology aims to systematically design and construct novel biological systems that address energy, environment, and health issues. Herein, we describe the development of a synthetic genetic system, which comprises quorum sensing, killing, and lysing devices, that enables Escherichia coli to sense and kill a pathogenic Pseudomonas aeruginosa strain through the production and release of pyocin. The sensing, killing, and lysing devices were characterized to elucidate their detection, antimicrobial and pyocin release functionalities, which subsequently aided in the construction of the final system and the verification of its designed behavior. We demonstrated that our engineered E. coli sensed and killed planktonic P. aeruginosa, evidenced by 99% reduction in the viable cells. Moreover, we showed that our engineered E. coli inhibited the formation of P. aeruginosa biofilm by close to 90%, leading to much sparser and thinner biofilm matrices. These results suggest that E. coli carrying our synthetic genetic system may provide a novel synthetic biology‐driven antimicrobial strategy that could potentially be applied to fighting P. aeruginosa and other infectious pathogens.     

Expression and glycosylation studies of human FGF receptor 4

Fibroblast growth factor receptor subtype 4 (FGFR4) has been shown to have special activation properties and just one splicing form, unlike the other FGFRs. FGFR4 overexpression is correlated with breast cancer and therefore FGFR4 is a target for drug design. Our aim is to overexpress high amounts of homogeneous FGFR4 extracellular domain (FGFR4(ed)) for structural studies. We show that baculovirus-insect cell-expressed FGFR4(ed) is glycosylated on three (N88, N234, and N266) of the six possible N-glycosylation sites but is not O-glycosylated. The deglycosylated triple mutant was expressed and had binding properties similar to those of glycosylated FGFR4(ed), but was still heterogeneous. Large amounts of FGFR4(ed) have been produced into inclusion bodies in Escherichia coli and refolded at least partly correctly but the refolded E. coli-produced FGFR4(ed) still aggregates.