Rolling circle amplification-based detection of human topoisomerase I activity on magnetic beads

A high-sensitivity assay has been developed for the detection of human topoisomerase I with single molecule resolution. The method uses magnetic sepharose beads to concentrate rolling circle products, produced by the amplification of DNA molecules circularized by topoisomerase I and detectable with a confocal microscope as single and discrete dots, once reacted with fluorescent probes. Each dot, corresponding to a single cleavage–religation event mediated by the enzyme, can be counted due to its high signal/noise ratio, allowing detection of 0.3 pM enzyme and representing a valid method to detect the enzyme activity in highly diluted samples.     

Hypoxia induces up-regulation of progranulin in neuroblastoma cell lines

Progranulin (PGRN) is a widely expressed multifunctional protein, involved in regulation of cell growth and cell cycle progression with a possible involvement in neurodegeneration. We looked for PGRN regulation in three different human neuroblastoma cell lines, following exposure to two different stimuli commonly associated to neurodegeneration: hypoxia and oxidative stress. For gene and protein expression analysis we carried out a quantitative RT-PCR and western blotting analysis. We show that PGRN is strongly up-regulated by hypoxia, through the mitogen-actived protein kinase (MAPK)/extracellular signal-regulated kinase (MEK) signaling cascade. PGRN is not up-regulated by H(2)O(2)-induced oxidative stress. These results suggest that PGRN in the brain could exert a protective role against hypoxic stress, one of principal risk factors involved in frontotemporal dementia pathogenesis.     

ADAM10 is the physiologically relevant, constitutive ��-secretase of the amyloid precursor protein in primary neurons

The amyloid precursor protein (APP) undergoes constitutive shedding by a protease activity called α‐secretase. This is considered an important mechanism preventing the generation of the Alzheimer's disease amyloid‐β peptide (Aβ). α‐Secretase appears to be a metalloprotease of the ADAM family, but its identity remains to be established. Using a novel α‐secretase‐cleavage site‐specific antibody, we found that RNAi‐mediated knockdown of ADAM10, but surprisingly not of ADAM9 or 17, completely suppressed APP α‐secretase cleavage in different cell lines and in primary murine neurons. Other proteases were not able to compensate for this loss of α‐cleavage. This finding was further confirmed by mass‐spectrometric detection of APP‐cleavage fragments. Surprisingly, in different cell lines, the reduction of α‐secretase cleavage was not paralleled by a corresponding increase in the Aβ‐generating β‐secretase cleavage, revealing that both proteases do not always compete for APP as a substrate. Instead, our data suggest a novel pathway for APP processing, in which ADAM10 can partially compete with γ‐secretase for the cleavage of a C‐terminal APP fragment generated by β‐secretase. We conclude that ADAM10 is the physiologically relevant, constitutive α‐secretase of APP.     

Automatic detection of surface EMG activation timing using a wavelet transform based method

The problem of the identification of the muscle contraction timing by using surface electromyographic signal is addressed. The timing detection of the muscular activation in dynamic conditions has a real clinical diagnostic impact. Widely used single threshold methods still rely on the experience of the operator in manually setting that threshold. A new approach to detect the muscular activation intervals, that is based on discontinuities detection in the wavelet domain, is proposed. Accuracy and precision of the algorithm were assessed by using a set of simulated signals obtaining values lower than 11.0 and 8.7 ms for biases and standard deviations of the estimation, respectively. Moreover an experimental application of the algorithm was carried out recruiting a population of 10 able-bodied subjects and processing the myoelectric signals recorded from the lower limb during an isokinetic exercise. The algorithm was able to reveal correctly the timing of muscular activation with performance comparable to the state-of-the-art methods. The detection algorithm is automatic and user-independent, it manages the detection of both onset and offset activation, it can be fruitfully applied even in presence of noise and, therefore, it can be used also by unskilled operators.     

UDP-GlC:glycoprotein glucosyltransferase-glucosidase II,the ying-yang of the ER quality control

The N-glycan-dependent quality control of glycoprotein folding prevents endoplasmic to Golgi exit of folding intermediates, irreparably misfolded glycoproteins and incompletely assembled multimeric complexes. It also enhances folding efficiency by preventing aggregation and facilitating formation of proper disulfide bonds. The control mechanism essentially involves four components, resident lectin-chaperones that recognize monoglucosylated polymannose glycans, a lectin-associated oxidoreductase acting on monoglucosylated glycoproteins, a glucosyltransferase that creates monoglucosytlated epitopes in protein-linked glycans and a glucosidase that removes the glucose units added by the glucosyltransferase. This last enzyme is the only mechanism component sensing glycoprotein conformations as it creates monoglucosylated glycans exclusively in not properly folded species or in not completely assembled complexes. The glucosidase is a dimeric heterodimer composed of a catalytic subunit and an additional one that is partially responsible for the ER localization of the enzyme and for the enhancement of the deglucosylation rate as its mannose 6-phosphate receptor homologous domain presents the substrate to the catalytic site. This review deals with our present knowledge on the glucosyltransferase and the glucosidase.     

Carbonic anhydrase inhibitors. Antioxidant polyphenols effectively inhibit mammalian isoforms I–XV

A series of polyphenolic derivatives, including resveratrol, dobutamine, curcumin, catechin and silymarine were investigated for the inhibition of all the catalytically active mammalian isozymes of the metalloprotein carbonic anhydrase (CA, EC 4.2.1.1), that is, CA I–CA XV. These polyphenols effectively inhibited CAs, with K_Is in the range of 380 nM–12.02 μM. The various isozymes showed quite diverse inhibition profiles with these compounds, which possess scaffolds not present in other investigated CA inhibitors (CAIs). These data may lead to drug design campaigns of effective CAIs possessing a diverse inhibition mechanism compared to sulfonamide/sulfamate inhibitors, based on such less investigated scaffolds.     

A Survey of Phytotoxic Microbial and Plant Metabolites as Potential Natural Products for Pest Management

Phytotoxic microbial metabolites produced by certain phytopathogenic fungi and bacteria, and a group of phytotoxic plant metabolites including Amaryllidacea alkaloids and some derivatives of these compounds were evaluated for algicide, bactericide, insecticide, fungicide, and herbicide activities in order to discover natural compounds for potential use in the management and control of several important agricultural and household structural pests. Among the various compounds evaluated: i) ophiobolin A was found to be the most promising for potential use as a selective algicide; ii) ungeremine was discovered to be bactericidal against certain species of fish pathogenic bacteria; iii) cycasin caused significant mortality in termites; iv) cavoxin, ophiobolin A, and sphaeropsidin A were most active towards species of plant pathogenic fungi; and v) lycorine and some of its analogues (1‐O‐acetyllycorine and lycorine chlorohydrate) were highly phytotoxic in the herbicide bioassay. Our results further demonstrated that plants and microbes can provide a diverse and natural source of compounds with potential use as pesticides.