Glutathione metabolism in Candida albicans resistant strains to fluconazole and micafungin.

Currently available therapies for candidiasis are based on antifungal drugs belonging to azole and echinocandin families that interfere with different aspects of fungal metabolism. These drugs, beyond their specific effects, elicit also a cellular stress including an unbalance of redox state that is counteracted not only utilizing antioxidant species but also increasing the outcome export by transporters to detoxify the internal environment. These cellular actions are both based on the cytosolic concentration of reduced glutathione (GSH). In this paper we investigated the effects of two antifungal drugs fluconazole and micafungin on the redox state of the cell in C. albicans to understand if the resistance to these drugs is accompanied by variation of glutathione metabolism. Analyses of resistant strains showed a marked difference in glutathione contents in strains resistant to fluconazole (CO23RFLC) or micafungin (CO23RFK). In CO23RFLC, the total amount of glutathione was more than doubled with respect to CO23RFK thanks to the increased activity of γ-glutamilcysteine synthetase, the key enzyme involved in GSH synthesis. We demonstrated that the GSH increase in CO23RFLC conferred to this strain a clear advantage in counteracting oxidative toxic agents while assignment of other roles, such as a more efficient elimination of the drug from the cell, should be considered more speculative. As far as MCFG resistance is concerned, from our data a role of glutathione metabolism in supporting this condition is not evident. Overall our data indicate that glutathione metabolism is differently affected in the two resistant strains and that glutathione system may play an important role in the global organization of C.albicans cells for resistance to fluconazole. Such scenario may pave the way to hypothesize the use of oxidant drugs or inhibitors able to deplete reduced glutathione level as a novel approach, for counteracting the resistance to this specific antifungal drug.

Cystamine restores GSTA3 levels in Vanin-1 null mice.

Free cysteamine levels in mouse tissues have been strictly correlated to the presence of membrane-bound pantetheinase activity encoded by Vanin-1. Vanin-1 is involved in many biological processes in mouse, from thymus homing to sexual development. Vanin-1 -/- mice are fertile and grow and develop normally; they better control inflammation and most of the knockout effects were rescued by cystamine treatment. Gene structure analysis showed the presence of an oxidative stimuli-responsive ARE-like sequence in the promoter. In this paper we investigate antioxidant-detoxifying enzymatic activities at the tissue level, comparing Vanin-1 -/- and wild-type mice. In Vanin-1 null animals we pointed out a decrease in the Se-independent glutathione peroxidase activity. The decrease in enzymatic activity appeared to be correlated to an impairment of GST isoenzyme levels. In particular a significant drop in GSTA3 together with a minor decrement in GSTM1 and an increase in GSTP1 levels was detected in Vanin-1 -/- livers. Cystamine administration to Vanin-1 -/- mice restored specifically GSTA3 levels and the corresponding enzymatic activity without influencing protein expression. A possible role of cystamine on protein stability/folding can be postulated.

Synthesis and characterization of a dehydrogenation product arising from the oxidation of aminoethylcysteine ketimine decarboxylated dimer.

While investigating the antioxidant properties of aminoethylcysteine ketimine decarboxylated dimer (1) (a natural substance occurring in biological fluids such as human urine and plasma and in bovine cerebellum), a previously unreported oxidation product was obtained. This compound was identified and characterized through comparison with an authentic sample prepared via Pd-catalyzed dehydrogenation of 1. This molecule is an example of an alternative oxidation pathway involving 1.

Glutathione S-transferase tissue profiling by reporter peptide monitoring.

Glutathione S-transferases (GSTs) form a widespread enzyme superfamily mainly involved in phase II detoxification. Differential expression of the various GST isoforms, differing in catalytic and structural properties, correlates with physiological and pathological states. Fast and simple determination of the GST profile is expected to be an important diagnostic tool in disease analysis. Here we propose a combined approach of high resolution separation techniques and electrospray mass spectrometric analyses for characterizing the spectrum of GSTs in male mouse liver. In this approach, the sensitivity and speed required for tissue GST profiling studies is achieved by tracking the reconstructed ion current of selected reporter peptides following chromatographic separation. This simple procedure, in which an affinity protein bait is followed by a chemical fragmentation and mass spectrometric analysis, could be sufficiently sensitive to detect the qualitative differences between physiological and pathological states.

[Measurement of sulfhemoglobin (S-Hb) blood levels to determine individual hydrogen sulfide exposure in thermal baths in Italy]. / La concentrazione di solfo-emoglobina presente nel sangue è una misura affidabile dell'esposizione individuale a vapori solfurei: il caso degli stabilimenti idrotermali in Italia.

Significant exposure to hydrogen sulfide may occur in workers at sulphureous thermal baths. Work-related exposure to hydrogen sulfide may be shown by measuring sulfhemoglobin (S-Hb) blood levels. In this study we measured S-Hb blood levels in two groups of workers at two different thermal baths and compared these with hydrogen sulfide concentrations in the air of the two work environments. Our results show that blood S-Hb levels can be considered a reliable measure of individual exposure to hydrogen sulfide.

Effect of aminoethylcysteine ketimine decarboxylated dimer, a natural sulfur compound present in human plasma, on tert-butyl hydroperoxide-induced oxidative stress in human monocytic U937 cells.

Aminoethylcysteine ketimine decarboxylated dimer (AECK-DD) is a natural sulphur compound present in human plasma and urine and in mammalian brain. Recently, it has been detected in many common dietary vegetables. The aim of the present study was to evaluate the ability of AECK-DD to affect cellular response of U937 human monocytic cells to tert-butyl hydroperoxide-induced oxidative stress. AECK-DD was incorporated into cells, as confirmed by GC-MS analyses, without any cytotoxic effect. A 24 h treatment with 50 and 250 microM AECK-DD resulted in the incorporation of 0.10 +/- 0.01 and 0.47 +/- 0.08ng AECK-DD x 10(6) cells, respectively. U937 cells pretreated with AECK-DD (in the range 4-100 microM) showed an increased resistance to tert-butyl hydroperoxide-induced necrotic death, as revealed by a higher percent of survival measured at all incubation times with respect to control cells. Moreover, the protective effect exhibited by AECK-DD is significantly stronger with respect to that obtained with other common antioxidants (N-acetyl cysteine and trolox) and comparable, although somewhat higher, to that of vitamin E. This effect seems to be due to the ability of AECK-DD to reduce glutathione depletion and to inhibit lipid peroxidation during tert-butyl hydroperoxide treatment. It can be concluded that AECK-DD protects cultured human monocytic cells against tert-butyl hydroperoxide-induced oxidative stress and subsequent cell death, likely through an antioxidant action inside the cell. Due to its presence in both human plasma and urine, AECK-DD may play a role in the modulation of oxidative processes in vivo.

Antioxidant properties of sulfinates: protective effect of hypotaurine on peroxynitrite-dependent damage.

It has been proposed that hypotaurine may function as an antioxidant in vivo. We investigated whether this compound can act as protective agent able to prevent damage from peroxynitrite, a strong oxidizing and nitrating agent that reacts with several biomolecules. The results showed that the compound efficiently protects tyrosine against nitration, alpha1-antiproteinase against inactivation, and human low-density lipoprotein against modification by peroxynitrite. Hypotaurine is also highly effective in inhibiting peroxynitrite-mediated nitration of tyrosine in the presence of added bicarbonate. This result suggests that hypotaurine could play an important role as protective agent under physiological conditions. Moreover, it was found that cysteine sulfinic acid, but not taurine, possesses protective properties against peroxynitrite-dependent damage similar to hypotaurine. These findings indicate that the protective effects exerted by these compounds may be attributable to the presence of the sulfinic group oxidizable into sulfonate by scavenging peroxynitrite and/or its derived species.

Synthesis and biological evaluation of the disulfide form of the glutathione analogue gamma-(L-glutamyl)-L-cysteinyl-L-aspartyl-L-cysteine.

By using the chain to chain mode of cyclization the title glutathione analogue (4), containing the 11-membered disulfide ring replacing the native -Cys-Gly fragment, has been synthesized and characterized together with its reduced dithiol form gamma-Glu-Cys-Asp-Cys (5). The activity of (4) with gamma-glutamyl-transferase and glutathione reductase has been evaluated and compared with those of the two conformationally restricted glutathione analogues (2) and (3) previously reported.

Proline-glutamate chimeras in isopeptides. Synthesis and biological evaluation of conformationally restricted glutathione analogues.

The two novel diastereoisomeric glutathione analogues 1 and 2 have been designed and synthesized by replacing the native gamma-glutamylic moiety with the conformational rigid skeleton of cis- or trans-4-carboxy-L-proline residue. Both analogues have been obtained by following the solution phase peptide chemistry methodologies and final reduction of the corresponding disulfide forms 13 and 14. The two analogues 1 and 2 have been tested towards gamma-glutamyltranspeptidase (gamma-GT) and human glutathione S-transferase (hGST P1-1). Both analogues 1 and 2 are completely resistant to enzymatic degradation by gamma-GT. The S-transferase utilizes the analogue 2 as a good substrate while is unable to bind the analogue 1.