Multiplexed reporter gene assays: monitoring the cell viability and the compound kinetics on luciferase activity

High-throughput screening assays with multiple readouts enable one to monitor multiple assay parameters. By capturing as much information about the underlying biology as possible, the detection of true actives can be improved. This report describes an extension to standard luciferase reporter gene assays that enables multiple parameters to be monitored from each sample. The report describes multiplexing luciferase assays with an orthogonal readout monitoring cell viability using reduction of resazurin. In addition, this technical note shows that by using the luciferin substrate in live cells, an assay time course can be recorded. This enables the identification of nonactive or unspecific compounds that act by inhibiting luciferase, as well as compounds altering gene expression or cell growth.     

Inhibition of neutrophil oxidative burst and granule secretion by Wortmannin: Potential role of MAP kinase and renaturable kinases

Exposure of neutrophils to a variety of agonists including soluble chemoattractant peptides and cytokines results in degranulation and activation of the oxidative burst (effector functions) that are required for bacterial killing. At present, the signaling pathways regulating these important functions are incompletely characterized. Mitogen-activated protein (MAP) kinases (MAPK) as well as members of a family of “renaturable kinases” are rapidly activated in neutrophils in response to diverse physiological agonists, suggesting that they may regulate cell activation. Antagonists of phosphatidyl inositol-3-(OH) kinase (PI3-kinase) such as wortmannin (Wtmn) inhibit these effector responses as well as certain of the above-mentioned kinases, leading to the suggestion that these enzymes lie downstream of PI3-kinase in the pathway regulating the oxidative burst and granule secretion. However, an apparent discrepancy exists in that, while virtually obliterating activity of PI3-kinase and the oxidase at low concentrations (ID₅₀ < 20 nM), Wtmn has only variable inhibitory effects on MAPK even at substantially higher concentrations (75–100 nM). This raises the possibility that the inhibitory effects of Wtmn are mediated via other enzyme systems. The purpose of the current study was therefore to compare the effects of Wtmn on PI3-kinase activity and on the chemoattractant-activated kinases, and to determine the potential relationship of these pathways to microbicidal responses. In human neutrophils, both the oxidative burst and granule secretion induced by fMLP were inhibited by Wtmn but at markedly different concentrations: the oxidative burst was inhibited with an ID₅₀ of <5 nM while granule secretion was only partially inhibited at concentrations exceeding 75 nM. Activation of both MEK-1 and MAPK in response to fMLP was only partially inhibited by high doses of Wtmn (ID₅₀ of > 100 nM and ≡75 nM, respectively). In contrast, Wtmn potently inhibited fMLP-induced activation of the 63 and 69 kDa renaturable kinases (ID₅₀ ≡ 5–10 nM). We speculate that the renaturable kinases may be involved in the regulation of the oxidative burst, whereas the MAPK pathway may play a role in other neutrophil functions such as granule secretion. J. Cell. Physiol. 172:94–108, 1997. © 1997 Wiley-Liss, Inc.     

Low sodium isocyanurate concentrations as a substitute to medium autoclaving in plant tissue culture

Plant Cell, Tissue and Organ Culture (PCTOC) - An optimization for a medium sterilization method, capable of substituting autoclaving, was developed using low concentrations of sodium isocyanurate...     

Carbohydrate metabolism and fruit quality are affected in frost-exposed Valencia orange fruit

Exposure of citrus fruit to frost often results in the development of freeze injury during their maturation in planta. This work was aimed to analyze changes in the biochemistry and enzymology of carbohydrate metabolism in freeze-injured orange fruit ( Citrus sinensis var. Valencia late) and the involvement of oxidative stress in frost damage. The activities of pyrophosphate-dependent phosphofructokinase, phosphoenolpyruvate carboxylase and several fermentative enzymes increased in frost-exposed (FE) fruit, while NADP-malic enzyme and the mitochondrial isoform of NAD-malate dehydrogenase showed a reduction in their activities. Western blot analysis indicated a correlation between activity levels and protein content. Respiration rate in whole fruit was reduced by 40%, whereas the flavedo showed a more pronounced decline (53%). Volatile compound (i.e. ethanol and acetaldehyde) content was significantly higher in FE fruit than in control, as was that of l -malate (three-fold). Additionally, FE fruit showed a marked decrease in the maturity index (24%) because of a higher titratable acidity (39%). Evidence is presented that oxidative stress is involved in freeze-induced damage of orange fruit, where oxidative damage to lipids and proteins, and a greater electrolyte leakage in the flavedo were also observed. The results suggest that freezing temperatures provoke a notable metabolic switch in citrus fruit toward a fermentative stage, resulting in low-quality fruits.     

Phagosomes fuse with late endosomes and/or lysosomes by extension of membrane protrusions along microtubules: role of Rab7 and RILP

Nascent phagosomes must undergo a series of fusion and fission reactions to acquire the microbicidal properties required for the innate immune response. Here we demonstrate that this maturation process involves the GTPase Rab7. Rab7 recruitment to phagosomes was found to precede and to be essential for their fusion with late endosomes and/or lysosomes. Active Rab7 on the phagosomal membrane associates with the effector protein RILP (Rab7-interacting lysosomal protein), which in turn bridges phagosomes with dynein-dynactin, a microtubule-associated motor complex. The motors not only displace phagosomes in the centripetal direction but, strikingly, promote the extension of phagosomal tubules toward late endocytic compartments. Fusion of tubules with these organelles was documented by fluorescence and electron microscopy. Tubule extension and fusion with late endosomes and/or lysosomes were prevented by expression of a truncated form of RILP lacking the dynein-dynactin-recruiting domain. We conclude that full maturation of phagosomes requires the retrograde emission of tubular extensions, which are generated by activation of Rab7, recruitment of RILP, and consequent association of phagosomes with microtubule-associated motors.     

The thermophilic biomass-degrading fungus Thielavia terrestris Co3Bag1 produces a hyperthermophilic and thermostable β-1,4-xylanase with exo- and endo-activity

Extremophiles - A hyperthermophilic and thermostable xylanase of 82&nbsp;kDa (TtXynA) was purified from the culture supernatant of T. terrestris Co3Bag1, grown on carboxymethyl cellulose (CMC),...     

Galactose-substituted alginate: preliminary characterization and study of gelling properties

Coupling of alginate with 1-amino-1-deoxygalactose in the presence of 1-ethyl-3-[3-(dimethylamino)propyl]carbodiimide results in a substituted polymer containing galactose side linked via an amide bond. To clarify the degree and pattern of substitution, a (1)H NMR study on the anomeric region of modified alginate, polymannuronate, alginate enriched in guluronic acid (G-enriched alginate), and polyalternating MG, was carried out (G, alpha-l-guluronic acid; M, beta-d-mannuronic acid). From the resonance of the proton at position 1 of galactosylamine, it was possible to determine the amount of galactose linked to mannuronic and to guluronic residues, respectively. Furthermore, (1)H NMR spectroscopy revealed a higher reactivity of guluronic residues for low degrees of conversion. Modified alginates with 7% and 19% of substitution are both able to form stable beads in the presence of calcium ions. The effect of galactose substitution on the dimensions, swelling, and stability of the beads has been studied and the cytotoxicity of the modified polymer evaluated in preliminary biological tests.