Anomaly Detection for Vision-based Railway Inspection

none7nomixedRiccardo Gasparini; Stefano Pini; Guido Borghi; Giuseppe Scaglione; Simone Calderara; Eugenio Fedeli; Rita CucchiaraRiccardo Gasparini; Stefano Pini; Guido Borghi; Giuseppe Scaglione; Simone Calderara; Eugenio Fedeli; Rita Cucchiar

IS THERE AN INTER-ORGAN GLUTATHIONE REDOX CYCLE

Intracellular glutathione redox status is a function of the flux through glutathione peroxidase-glutathione reductase system. Specific activities of these two enzymes in rat liver cytosol and erythrocyte hemolysates were determined. Relative to glutathione peroxidase levels, glutathione reductase activity was about 15-fold more abundant in the rat liver than in erythrocytes. This is suggestive of greater capacity of the liver to reduce oxidised glutathione (GSSG). Based on these results and from the pattern of glutathione efflux from different cells and tissues [Sies, H. & Akerboom, T.P.M. (1984) Methods Enzymol. 105, 445-451], it is speculated that an interorgan glutathione redox cycle may be operative wherein liver is central to the reduction of GSSG and other disulphides

A simple setup to illustrate metabolic pathway dynamics

The sense of dynamic nature of a metabolic pathway, particularly in its steady state, is the most difficult to convey to the student. Such insights into metabolism have come from extensive and cumulative experimental data. These are not easily amenable to laboratory demonstrations. A working model is described here to illustrate metabolic concepts in a laboratory and to complement classroom teaching. (C) 2000 IUBMB. .

Phosphinothricin resistance in Aspergillus niger and its utility as a selectable transformation marker

Aspergillus niger is moderately susceptible to inhibition by phosphinothricin (PPT)-a potent inhibitor of glutamine synthetase. This growth inhibition was relieved by L-glutamine. PPT inhibited A. niger glutamine synthetase in vitro (K-I, 54 mu M) and the inhibition was competitive with L-glutamate. The bar gene, imparting resistance to PPT, was successfully exploited as a dominant marker to transform this fungus. Very high PPT concentrations were required in the overlay for selection. Apart from bar transformants, colonies spontaneously resistant to PPT were frequently encountered on selection media. Reasons for such spontaneous resistance, albeit of moderate growth phenotype, were sought using one such isolate (SRPPT). The SRPPT isolate showed a 2-3-fold decrease in its glutamate uptake rate. Elevated external glutamate levels further suppressed the PPT-induced growth inhibition. Cellular entry of PPT could be through the L-glutamate uptake system thereby accounting for the observed spontaneous resistant phenotype. These results were useful in the fine-tuning of bar-selection in A. niger. (C) 200

The metabolism of 4-aminobutyrate (GABA) in fungi

Information on the genetics and metabolism of 4-aminobutyrate (GABA) in yeasts and fungi is reviewed. In spite of ubiquitous occurrence, there is limited information on its function and biological role. Most fungi utilize GABA both as a carbon and a nitrogen source. Fungal endogenous GABA largely originates from the decarboxylation of L-glutamate and is associated with sporulation/spore metabolism. Whatever its source, GABA is catabolized to succinate via succinicsemialdehyde. Taken together these steps define a potential bypass outside the classical tricarboxylic acid cycle. Evidence for the existence of such a functional bypass in fungi is reviewed. The role of GABA and its metabolism in various facets of fungal biology is gradually emerging

Utility of Aspergillus niger citrate synthase promoter for heterologous expression

Citrate synthase is a central player in the acidogenic metabolism of Aspergillus niger. The 5' upstream sequence (0.9 kb DNA) of citrate synthase gene (citA) from A. niger NCIM 565 was analyzed and its promoter function demonstrated through the heterologous expression of two proteins. The cloned citrate synthase promoter (PcitA) sequence was able to express bar coding sequence thereby conferring phosphinothricin resistance. This sequence was further analyzed by systematic deletions to define an effective but compact functional promoter. The PcitA driven egfp expression showed that PcitA was active in all differentiation cell-stages of A. niger. EGFP expression was highest on non-repressible carbon sources like acetate and glycerol. Mycelial EGFP levels increased during acidogenic growth suggesting that PcitA is functional throughout this cultivation. A. niger PcitA is the first Krebs cycle gene promoter used to express heterologous proteins in filamentous fungi. (C) 2011 Elsevier B.V. All rights reserved

Cloning and use of sC as homologous marker for Aspergillus niger transformation

The sC sequence from Aspergillus niger was cloned and developed into a homologous marker system for genetic transformation. The coding region of the sC gene amplified by PCR from the A. niger genome was provided with Aspergillus nidulans expression signals (gpdA promoter and trpC terminator). This chimeric construct was used to successfully transform a spontaneous sC(-) isolate of A. niger to prototrophy. The transformants analyzed by Southern analysis showed integration of multiple copies of the transforming DNA. They also exhibited much higher ATP sulfurylase activity than the wild-type A. niger strain reinforcing the molecular data. This demonstrates the usefulness of the sC(niger) construct, driven by PgpdA, as a marker for A. niger transformation. (C) 200

Genetic transformation in aspergilli: tools of the trade

DNA-mediated transformation is a powerful tool that allows the introduction of specific genetic changes in an organism. Transformation of Aspergilli, acclaimed for their wide use in the industry, has been possible for about two decades now. Several basic and applied problems related to fungal biology have been addressed using this technique. Nonetheless, new markers and strategies for transformation are still being developed for these filamentous fungi. Different methods and markers that are currently available for the transformation of Aspergilli are summarized here. The review also brings out the importance of these transformation systems in analyzing fungal gene function. Aspects of Aspergillus niger transformation are selectively emphasized

Allosteric NADP-glutamate dehydrogenase from aspergilli: purification, characterization and implications for metabolic regulation at the carbon-nitrogen interface

NADP-dependent glutamate dehydrogenase (NADP-GDH) mediates fungal ammonium assimilation through reductive synthesis of glutamate from 2-oxoglutarate. By virtue of its position at the interface of carbon and nitrogen metabolism, biosynthetic NADP-GDH is a potential candidate for metabolic control. In order to facilitate characterization, a new and effective dye-affinity method was devised to purify NADP-GDH from two aspergilli, Aspergillus niger and Aspergillus nidulans. The A. niger NADP-GDH was characterized at length and its kinetic interaction constants with glutamate (K-m 34(.)7 mM) and ammonium (K-m 1(.)05 mM; K-i 0(.)4 mM) were consistent with an anabolic role. Isophthalate, 2-methyleneglutarate and 2,4-pyridinedicarboxylate were significant inhibitors, with respective K-i values of 6(.)9, 9(.)2 and 202(.)0 mu M. The A. niger enzyme showed allosteric properties and a sigmoid response (n(H) = 2(.)5) towards 2-oxoglutarate saturation. The co-operative behaviour was a feature common to NADP-GDH from Aspergillus awamori, A. nidulans and Aspergillus oryzae. NADP-GDH may therefore be a crucial determinant in adjusting 2-oxoglutarate flux between the tricarboxylic acid cycle and glutamate biosynthesis in aspergilli