Silencing of mitochondrial alternative oxidase gene of <Emphasis Type="Italic">Aspergillus fumigatus</Emphasis> enhances reactive oxygen species production and killing of the fungus by macrophages

We previously demonstrated that conidia from Aspergillus fumigatus incubated with menadione and paraquat increases activity and expression of cyanide-insensitive alternative oxidase (AOX). Here, we employed the RNA silencing technique in A. fumigatus using the vector pALB1/aoxAf in order to down-regulate the aox gene. Positive transformants for aox gene silencing of A. fumigatus were more susceptible both to an imposed in vitro oxidative stress condition and to macrophages killing, suggesting that AOX is required for the A. fumigatus pathogenicity, mainly for the survival of the fungus conidia during host infection and resistance to reactive oxygen species generated by macrophages.     

Exosome biogenesis,bioactivities and functions as new delivery systems of natural compounds

A rapidly growing body of experimental evidence has begun to shed light on the wide ranging molecular mechanisms which modulate intra- and inter-cellular communications. A substantial quantity of the available knowledge has only been uncovered in recent years, and we are learning that donor cells release nanovesicles, known as exosomes, which regulate the cellular behavior of recipient cells following uptake. Based on the impressive capacity of exosomes in delivering their “payload”, different therapeutic agents, are currently being tested using this delivery method for more effective therapy. This review summarizes the most recent developments in exosome bioactivities and discusses the biochemical nature of exosomes and their biogenesis. It also summarizes the use of exosomes as delivery vehicles for drugs and natural compounds to the targeted site.     

Synergistic activation of plasma membrane H+– ATPase in Arabidopsis thaliana cells by turgor decrease and by fusicoccin

The regulation of the H⁺-ATPase of plasma membrane is a crucial point in the integration of transport processes at this membrane. In this work the regulation of H⁺-ATPase activity induced by changes in turgor pressure was investigated and compared with the stimulating effect of fusicoccin (FC). The exposure of cultured cells of Arabidopsis thaliana L. (ecotype Landsberg 310–14-2) to media containing mannitol (0. 15 or 0. 3 M ) or polyethylene glycol 6000 (PEG) (15. 6% or 22% w/v) resulted in a decrease in the turgor pressure of the cells and in a strong stimulation of H⁺ extrusion in the incubation medium. The osmotica-induced H⁺ extrusion was (1) inhibited by the inhibitor of plasma membrane H⁺-ATPase, erythrosin B (EB), (2) dependent on the external K⁺ concentration, (3) associated with a net K⁺ influx, and (4) lead to an increase of cellular malate content. These results show that the reduction of external osmotic potential stimulates the activity of plasma membrane H⁺-ATPase
The effect of mannitol was only partially inhibited by treatments with cycloheximide (CH) and cordycepin, which block protein and mRNA synthesis, respectively. All the effects of osmotica were qualitatively and quantitatively similar to those induced by 5 μ M FC. However, when FC and mannitol (or PEG) were fed together, their effects on H⁺ extrusion appeared synergistic, irrespective of whether FC was present at suboptimal or optimal concentrations. This behaviour suggests that the modes of action of FC and of the osmotica on H⁺-ATPase activity differ at least in some step(s)     

Effect of a ubiquinone-like molecule on oxidative energy metabolism in rat cortical synaptosomes at different ages

Persistent stimulation of energy consumption, induced by depolarization with veratridine, mimics a condition of abnormally enhanced energy demand and causes an increase in the oxygen consumption rate (QO₂) and in the interconversion of pyruvate dehydrogenase complex (PDHc) into its active form. Wistar rats at the age of 26 months do not show alterations of QO₂ and of the ability of veratridine to increase QO₂ in comparison with 6 month-old animals whereas the active form of PDHc is slightly but significantly reduced. Idebenone, a ubiquinone-like molecule (1 M), does not affect the QO₂ or PDHc activation state in resting conditions but attenuates the veratridine-challenged increase in QO₂ at all the ages tested and attenuates the increase in the percentage of PDHa reaching statistical significance in 26-month-old rats. At higher concentration (10 M) idebenone totally abolishes the veratridine-induced increase in PDHa also in the 6 month-old rats. At the lower concentration, the drug does not affect the increase in QO₂ induced by an uncoupler of oxidative phosphorylation. The results obtained suggest a protective effect of idebenone on the cerebral tissue against stressful conditions; this action may be exerted at the level of some mitochondrial component and/or on the Na⁺ homeostasis.     

The kinetic mechanism of the reactions catalyzed by the glutamate synthase from Azospirillum brasilense

The reactions catalyzed by glutamate synthase from Azospirillum brasilense have been investigated by a combination of absorption spectroscopy, steady-state kinetic measurements and experiments with stereospecifically labelled substrate. The data show that both L-glutamine-dependent and ammonia-dependent reactions of the glutamate synthase from A. brasilense follow an identical two-site uni-uni bi-bi kinetic mechanism, in which the enzyme is alternately reduced by NADPH and oxidized by the iminoglutarate formed on addition of ammonia to the C2 of 2-oxoglutarate. The spectroscopic experiments support the involvement of the enzyme chromophores (flavins and iron-sulfur centers) in both reactions. Finally, using stereospecifically labelled NADPH, we showed that the enzyme from Azospirillum is specific for the transfer of the 4S hydrogen of NADPH. During the catalysis of both L-glutamine-dependent and ammonia-dependent reactions, this hydrogen atom equilibrates with the solvent. The data obtained with glutamate synthase from A. brasilense, a diazotroph, differ significantly from those regarding the ammonia-dependent reaction of other glutamate synthases. The ammonia-dependent activity of glutamate synthase from Azospirillum is not physiologically significant, representing only a segment of the overall physiological L-glutamine-dependent activity and requiring the enzyme flavins and iron-sulfur centers. Finally, the data are not consistent with the hypothesis [Geary, L. E. & Meister, A. (1977) J. Biol. Chem. 252, 3501-3508] that the small subunit of glutamate synthase is endowed with a glutamate-dehydrogenase-like activity.     

Effect of Zn(II) and Mg(II) on phosphohydrolytic activity of rat matrix-induced alkaline phosphatase

Rat matrix-induced alkaline phosphatase is an enzyme which requires magnesium and zinc ions for its maximal activity. Two Zn(II) ions and one Mg(II) ion are bound to each subunit of native dimeric enzyme. The presence of magnesium ion (10-100 microM) or zinc ion (7-20 nM) alone is sufficient to stimulate apoenzyme activity. However maximal activity (264 U/mg) requires the presence of both ions. Binding of Zn(II) ions to the Mg(II) binding site causes a strong inhibition of the apoenzyme while the binding of Mg(II) on Zn(II) binding site is not sufficient to stimulate PNPPase activity of the apoenzyme. Binding of both ions to the enzyme molecule did not change the apparent dissociation constant for PNPP hydrolysis.     

Immunoelectron microscopic localization of D-amino acid oxidase in rat kidney and liver

Summary The intracellular localization ofd-amino acid oxidase in rat kidney and liver has been investigated using the indirect immunogold postembedding technique. Different fixation and embedding conditions for optimal preservation of antigenicity and fine structure have been tested. Immunolabelling was possible only in tissues embedded in polar resins (glycol methacrylate and Lowicryl K4M). In kidney the enzyme was demonstrable only in the peroxisomes of the proximal tubule, where it was associated with the peroxisome core. The enzyme was present in all the peroxisomes of the proximal tubule and appeared to be codistributed with catalase. Control experiments and quantitative analysis confirmed the specificity of thed-amino acid oxidase immunolocalization. All the other cells in kidney failed to demonstrate any labelling. In liver, the immunolabelling was present in the matrix of the hepatocyte peroxisomes, whereas no traces of the enzyme were found in the nucleoid. The intensity of the immunolabelling in liver peroxisomes was lower than in kidney. No specific labelling was observed in cells other than hepatocytes.     

Purification and characterization of glutamate synthase from Azospirillum brasilense.

Growth conditions for Azospirillum brasilense Sp6 were devised for maximal expression of glutamate synthase. The enzyme levels were largely affected by the type and concentration of the nitrogen source. A 10-fold increase in the synthesis of the enzyme was observed at a limiting concentration of ammonia. The enzyme was purified to homogeneity by a procedure which was fairly rapid and allowed a good recovery of enzyme (30%). Azospirillum glutamate synthase is a complex iron-sulfur flavoprotein with a stoichiometry of 1 flavin adenine dinucleotide:1 flavin mononucleotide:8 Fe:8 S per protomer with a molecular weight of 185,000. The protomer is composed of two dissimilar subunits with molecular weights of 135,000 and 50,000. Kinetic parameters were determined. Km values for NADPH, 2-oxoglutarate, and L-glutamine were 6.25, 29, and 450 microM, respectively. The optimum pH was about 7.5. Complete reduction of the enzyme under anaerobic conditions was obtained either by NADPH (in the presence of a regenerating system) or dithionite or by photochemical reduction (in the presence of EDTA and 5-deazariboflavin). No stable long-wavelength intermediates were observed.     

Juvenile active ossifying fibroma with massive involvement of the mandible

Fibro-osseous lesions of the maxillofacial complex are often difficult to diagnose from both a clinical and a histopathologic point of view. The parameters for the diagnosis of juvenile active ossifying fibroma are as follows: a patient under 15 years of age, localization of the tumor, the radiologic aspect, and the tendency to recur. Although many authors favor conservative surgery rather than radical en bloc resection, immediate recurrence characterized by a high aggressive growth rate and the absence of a distinct separation between the tumor and the adjacent bone requires ex- tensive surgery, with wide demolition of the involved bone.