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Autophagy is a mechanism responsible for intracellular degradation and recycling of macromolecules and organelles, essential for cell survival in adverse conditions. More than 40 autophagy-related (ATG) genes have been identified and characterized in fungi, among them ATG4 and ATG8. ATG4 encodes a cysteine protease (Atg4) that plays an important role in autophagy by initially processing Atg8 at its C-terminus region. Atg8 is a ubiquitin-like protein essential for the synthesis of the double-layer membrane that constitutes the autophagosome vesicle, responsible for delivering the cargo from the cytoplasm to the vacuole lumen. The contributions of Atg-related proteins in the pathogenic yeast in the genus Cryptococcus remain to be explored, to elucidate the molecular basis of the autophagy pathway. In this context, we aimed to investigate the role of autophagy-related proteins 4 and 8 (Atg4 and Atg8) during autophagy induction and their contribution with non-autophagic events in C. neoformans. We found that Atg4 and Atg8 are conserved proteins and that they interact physically with each other. ATG gene deletions resulted in cells sensitive to nitrogen starvation. ATG4 gene disruption affects Atg8 degradation and its translocation to the vacuole lumen, after autophagy induction. Both atg4 and atg8 mutants are more resistant to oxidative stress, have an impaired growth in the presence of the cell wall-perturbing agent Congo Red, and are sensitive to the proteasome inhibitor bortezomib (BTZ). By that, we conclude that in C. neoformans the autophagy-related proteins Atg4 and Atg8 play an important role in the autophagy pathway; which are required for autophagy regulation, maintenance of amino acid levels and cell adaptation to stressful conditions.

Recently, we showed that intravenous (i.v.) treatment with the essential oil of Mentha x villosa (EOMV) in pentobarbitone-anaesthetised rats decreased blood pressure; the effect occurred independently of the presence of an operational central autonomic drive to the cardiovascular system. This finding suggested that the hypotensive activity of EOMV may result from its vasodilatory effects directly upon vascular smooth muscle. The present study examines this possibility and whether EOMV-induced hypotension is mediated, at least in part, by an endothelial L-arginine/nitric oxide pathway. In conscious rats, i.v. injections of bolus doses (1 to 20 mg/kg) of EOMV elicited immediate and dose-dependent decreases in mean aortic pressure (MAP) and heart rate (HR). Pretreatment with i.v. hexamethonium (30 mg/kg) reduced the EOMV-induced bradycardia without affecting the hypotension. However, i.v. pretreatment with the nitric oxide synthase inhibitor, N(G)-nitro-L-arginine methyl (L-NAME, 20 mg/kg), reduced partially, but significantly, the maximal percent decreases in MAP elicited by EOMV without affecting the bradycardia. In rat isolated thoracic aorta preparations, EOMV (1 - 130 microg/ml) induced a concentration-dependent reduction of potassium (60 mM)-induced contraction. This smooth muscle-relaxant activity of EOMV was significantly reduced by the incubation of endothelium-intact rings with L-NAME (20 microM), as evidenced by the significant enhancement in the IC50 for EOMV-induced reduction of potassium-induced contraction (133.8 +/- 26.5 vs. 65.2 +/- 8.2 microg/ml in the absence of L-NAME). Furthermore, the vasorelaxant effects of EOMV in endothelium-denuded aortic rings were also significantly reduced (IC50 = 109 +/- 10 microg/ml), compared to those observed in segments with intact endothelium (IC50 = 61 +/- 13 microg/ml). These results show that i.v. treatment with EOMV dose-dependently decreases blood pressure in conscious rats, and that this action is due to an active vascular relaxation rather than withdrawal of sympathetic tone. Released nitric oxide from vascular endothelial cells appears partially involved in the aortic relaxation induced by EOMV and in turn in the mediation of EOMV-induced hypotension. They further support the concept that EOMV-induced hypotension and bradycardia occurred independently.

Cardiovascular effects of intravenous (i. v.) treatment with the essential oil of Mentha x villosa (EOMV) were investigated in pentobarbitone-anaesthetised rats. Additionally this study examines whether the major constituent of EOMV, piperitenone oxide (PO), is the active principle mediating EOMV-induced changes in mean aortic pressure (MAP) and heart rate (HR) and whether the autonomic nervous system is involved in the mediation of these cardiovascular effects. Two samples of EOMV have been tested: one contained 62.32% of PO (sample 1) and the other contained a higher percent (95.87%) of PO (sample 2). Intravenous injections of bolus doses (1 to 20 mg/kg) of both samples of EOMV elicited immediate and dose-dependent decreases in MAP and HR. These cardiovascular responses were also observed following i. v. injections of PO (1 to 20 mg/kg). However, maximal percent decreases in MAP and HR elicited by sample 2 of EOMV were significantly greater than those evoked by sample 1 of EOMV, while they were of the same order of magnitude as those elicited by PO. Pretreatment of rats with either bilateral vagotomy or i. v. methylatropine (1 mg/kg) did not modify significantly the hypotensive and bradycardic responses to EOMV. In contrast, pretreatment with i. v. hexamethonium (30 mg/kg) partially, but significantly, reduced the bradycardic effects of EOMV without affecting hypotension. The present study shows for the first time that i. v. treatment with EOMV in pentobarbitone-anaesthetised rats induces hypotensive and bradycardic effects, which appear mostly attributed to the actions of the major constituent of EOMV, PO. These cardiovascular effects appear to be independent since EOMV-induced bradycardia appears dependent upon the presence of an intact and functional sympathetic nerve drive to the heart, while EOMV-induced hypotension appears independent of the presence of an operational sympathetic nervous system. This suggests that hypotensive activity of EOMV may result from its vasodilatory effects directly upon vascular smooth muscle.