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Aging is a natural process characterized by several biological changes. In this context, oxidative stress appears as a key factor that leads cells and organisms to severe dysfunctions and diseases. To cope with reactive oxygen species and oxidative-related damage, there has been increased use of superoxide dismutase (SOD)/catalase (CAT) biomimetic compounds. Recently, we have shown that three metal-based compounds {[Fe(HPClNOL)Cl2]NO3, [Cu(HPClNOL)(CH3CN)](ClO4)2 and Mn(HPClNOL)(Cl)2}, harboring in vitro SOD and/or CAT activities, were critical for protection of yeast cells against oxidative stress. In this work, treating Saccharomyces cerevisiae with these SOD/CAT mimics (25.0 mM/1 h), we highlight the pivotal role of these compounds to extend the life span of yeast during chronological aging. Evaluating lipid and protein oxidation of aged cells, it becomes evident that these mimics extend the life expectancy of yeast mainly due to the reduction in oxidative stress biomarkers. In addition, the treatment of yeast cells with these mimics regulated the amounts of lipid droplet occurrence, consistent with the requirement and protection of lipids for cell integrity during aging. Concerning SOD/CAT mimics uptake, using inductively coupled plasma mass spectrometry, we add new evidence that these complexes, besides being bioabsorbed by S. cerevisiae cells, can also affect metal homeostasis. Finally, our work presents a new application for these SOD/CAT mimics, which demonstrate a great potential to be employed as antiaging agents. Taken together, these promising results prompt future studies concerning the relevance of administration of these molecules against the emerging aging-related diseases such as Parkinson's, Alzheimer's and Huntington's. [ABSTRACT FROM AUTHOR]

There is substantial interest in developing alternative strategies for cancer chemotherapy aiming to increase drug specificity and prevent tumor resistance. Calorie restriction (CR) has been shown to render human cancer cells more susceptible to drugs than normal cells. Indeed, deficiency of nutrient signaling proteins mimics CR, which is sufficient to improve oxidative stress response and life expectancy only in healthy cells. Thus, although CR and reduction of nutrient signaling may play an important role in cellular response to chemotherapy, the full underlying mechanisms are still not completely understood. Here, we investigate the relationship between the nutrient sensor proteins Ras2, Sch9, or Tor1 and the response of calorie-restricted Saccharomyces cerevisiae cells to cisplatin. Using wild-type and nutrient-sensing mutant strains, we show that deletion of any of these proteins mimics CR and is sufficient to increase cell protection. Moreover, we show that glutathione (GSH) is essential for proper CR protection of yeast cells under cisplatin chemotherapy. By measuring the survival rates and GSH levels, we found that cisplatin cytotoxicity leads to a decrease in GSH content reflecting in an increase of oxidative damage. Finally, investigating DNA fragmentation and apoptosis, we conclude that GSH contributes to CR-mediated cell survival. [ABSTRACT FROM AUTHOR]

The Ca2+-calcineurin signaling pathway in the human fungal pathogen Cryptococcus neoformans is essential for adaptation to the host environment during infection. Calcium transporters regulate cytosolic calcium concentrations, providing Ca2+ loading into storage organelles. The three calcium transporters that have been characterized in C. neoformans, Cch1, Eca1 and Vcx1, are required for fungal virulence, supporting a role for calcium-mediated signaling in cryptococcal pathogenesis. In the present study, we report the functional characterization of the putative vacuolar calcium ATPase Pmc1 in C. neoformans. Our results demonstrate that Pmc1 provides tolerance to high Ca2+ concentrations. The double knockout of C. neoformans PMC1 and VCX1 genes impaired the intracellular calcium transport, resulting in a significant increase in cytosolic calcium levels. Furthermore, Pmc1 was essential for both the progression of pulmonary infection and brain colonization in mice, emphasizing the crucial role of calcium signaling and transport for cryptococcal pathogenesis. [ABSTRACT FROM AUTHOR]

Capsule production is common among bacterial species, but relatively rare in eukaryotic microorganisms. Members of the fungal Cryptococcus genus are known to produce capsules, which are major determinants of virulence in the highly pathogenic species Cryptococcus neoformans and Cryptococcus gattii. Although the lack of virulence of many species of the Cryptococcus genus can be explained solely by the lack of mammalian thermotolerance, it is uncertain whether the capsules from these organisms are comparable to those of the pathogenic cryptococci. In this study, we compared the characteristic of the capsule from the non-pathogenic environmental yeast Cryptococcus liquefaciens with that of C. neoformans. Microscopic observations revealed that C. liquefaciens has a capsule visible in India ink preparations that was also efficiently labeled by three antibodies generated to specific C. neoformans capsular antigens. Capsular polysaccharides of C. liquefaciens were incorporated onto the cell surface of acapsular C. neoformans mutant cells. Polysaccharide composition determinations in combination with confocal microscopy revealed that C. liquefaciens capsule consisted of mannose, xylose, glucose, glucuronic acid, galactose and N-acetylglucosamine. Physical chemical analysis of the C. liquefaciens polysaccharides in comparison with C. neoformans samples revealed significant differences in viscosity, elastic properties and macromolecular structure parameters of polysaccharide solutions such as rigidity, effective diameter, zeta potential and molecular mass, which nevertheless appeared to be characteristics of linear polysaccharides that also comprise capsular polysaccharide of C. neoformans. The environmental yeast, however, showed enhanced susceptibility to the antimicrobial activity of the environmental phagocytes, suggesting that the C. liquefaciens capsular components are insufficient in protecting yeast cells against killing by amoeba. These results suggest that capsular structures in pathogenic Cryptococcus species and environmental species share similar features, but also manifest significant difference that could influence their potential to virulence. [ABSTRACT FROM AUTHOR]

Microplusin is an antimicrobial peptide isolated from the cattle tick R hipicephalus ( B oophilus) microplus. Its copper-chelating ability is putatively responsible for its bacteriostatic activity against M icrococcus luteus as microplusin inhibits respiration in this species, which is a copper-dependent process. Microplusin is also active against C ryptococcus neoformans ( MIC50 = 0.09 μM), the etiologic agent of cryptococcosis. Here, we show that microplusin is fungistatic to C . neoformans and this inhibitory effect is abrogated by copper supplementation. Notably, microplusin drastically altered the respiratory profile of C . neoformans. In addition, microplusin affects important virulence factors of this fungus. We observed that microplusin completely inhibited fungal melanization, and this effect correlates with the inhibition of the related enzyme laccase. Also, microplusin significantly inhibited the capsule size of C . neoformans. Our studies reveal, for the first time, a copper-chelating antimicrobial peptide that inhibits respiration and growth of C . neoformans and modifies two major virulence factors: melanization and formation of a polysaccharide capsule. These features suggest that microplusin, or other copper-chelation approaches, may be a promising therapeutic for cryptococcosis. [ABSTRACT FROM AUTHOR]

Melanin formation is a promising target for antifungal development. We screened a collection of 727 compounds that were previously approved for clinical use in humans for inhibition of pigmentation in Cryptococcus gattii, a lethal fungal pathogen that causes damage to both immunocompetent and immunocompromised hosts. The pyrimidine analogues flucytosine (5-fluorocytosine [5-FC]), 5-fluorouracil (5-FU) and carmofur were identified as efficient inhibitors of pigmentation in the C. gattii model. Since melanin synthesis is enzymatically catalyzed by laccase in Cryptococcus, we investigated whether inhibition of pigmentation by the pyrimidine analogues was laccase-mediated. Enzyme activity and expression of LAC genes were not involved in the effects of the pyrimidine analogues, suggesting alternative cellular targets for inhibition of pigmentation. To address this hypothesis, we screened a collection of approximately 8000 mutants of C. gattii that were produced by insertional mutation after incubation with Agrobacterium tumefaciens and identified a gene product required for the anti-pigmentation activity of 5-FC as a beta-DNA polymerase. Reduced expression of this gene affected capsule formation and urease activity, suggesting essential roles in the cryptococcal physiology. These results demonstrate a previously unknown antifungal activity of 5-FC and reveal a promising target for the development of novel antifungals. [ABSTRACT FROM AUTHOR]

Cryptococcus neoformans is an encapsulated yeast that causes a life-threatening meningoencephalitis in immunocompromised individuals. The ability to survive and proliferate at the human body temperature is an essential virulence attribute of this pathogen. This trait is controlled in part by the Ca²(+)-calcineurin pathway, which senses and utilizes cytosolic calcium for signaling. In the present study, the identification of the C. neoformans gene VCX1, which encodes a vacuolar calcium exchanger, is reported. The VCX1 knockout results in hypersensitivity to the calcineurin inhibitor cyclosporine A at 35°C, but not at 30°C. Furthermore, high concentrations of CaCl₂ lead to growth inhibition of the vcx1 mutant strain only in the presence of cyclosporine A, indicating that Vcx1 acts in parallel with calcineurin. The loss of VCX1 does not influence cell wall integrity or capsule size but decreases secretion of the major capsular polysaccharide glucuronoxylomannan (GXM) in culture supernatants.Vcx1 also influences C. neoformans phagocytosis by murine macrophages and is required for full virulence in mice. Analysis of cellular distribution by confocal microscopy confirmed the vacuolar localization of Vcx1 in C. neoformans cells.