Analysis of biological thiols: derivatization with monobromotrimethylammoniobimane and characterization by electrophoresis and chromatography

A new method for analysis of biological thiols based upon their conversion to fluorescent derivatives by reaction with monobromotrimethylammoniobimane (qBBr) is described. The derivatives are separated by chromatography and by electrophoresis on cellulose thinlayer chromatography plates. The use of two-dimensional mapping makes it possible to differentiate between a wide variety of biological thiols including N-acetylcysteine, CoA, cysteine, cysteinylglycine, cysteamine, ergothioneine, glutathione, γ-glutamylcysteine, homocysteine, mercaptopyrimidine, pantetheine, 4′-phosphopanetheine, thiosulfate, and thiouracil. For applications to biological samples thiols were isolated from crude extracts by binding to a mercuriagarose gel. Following removal from the gel with dithiothreitol, the thiols were derivatized with qBBr. The methods were tested by showing that glutathione is the major thiol in human red blood cells, that glutathione and ergothioneine are the major thiols in Neurospora crassa conidia, and that Bacillus cereus vegetative cells lack glutathione but contain cysteine, pantetheine, and an unidentified thiol in significant amounts.     

The Protective Effect of N-Acetylcysteine on Oxidative Stress in the Brain Caused by the Long-Term Intake of Aspartame by Rats

Long-term intake of aspartame at the acceptable daily dose causes oxidative stress in rodent brain mainly due to the dysregulation of glutathione (GSH) homeostasis. N-Acetylcysteine provides the cysteine that is required for the production of GSH, being effective in treating disorders associated with oxidative stress. We investigated the effects of N-acetylcysteine treatment (150 mg kg^?1, i.p.) on oxidative stress biomarkers in rat brain after chronic aspartame administration by gavage (40 mg kg^?1). N-Acetylcysteine led to a reduction in the thiobarbituric acid reactive substances, lipid hydroperoxides, and carbonyl protein levels, which were increased due to aspartame administration. N-Acetylcysteine also resulted in an elevation of superoxide dismutase, glutathione peroxidase, glutathione reductase activities, as well as non-protein thiols, and total reactive antioxidant potential levels, which were decreased after aspartame exposure. However, N-acetylcysteine was unable to reduce serum glucose levels, which were increased as a result of aspartame administration. Furthermore, catalase and glutathione S-transferase, whose activities were reduced due to aspartame treatment, remained decreased even after N-acetylcysteine exposure. In conclusion, N-acetylcysteine treatment may exert a protective effect against the oxidative damage in the brain, which was caused by the long-term consumption of the acceptable daily dose of aspartame by rats.     

Studies on the reactivity of the essential cysteine of thymidylate synthetase

The reaction of one of the four cysteinyl residues of thymidylate synthetase from methotrexate-resistant Lactobacillus casei with a variety of sulfhydryl reagents results in complete inhibition of the enzyme. Kinetic studies indicate that the rates of reactivity of the reagents tested are N-ethylmaleimide > iodoacetamide > N-(iodoacetylaminoethyl)-S-naphthylamine-1-sulfonic acid > iodoacetic acid. The enzyme is also inactivated by 5-Hg-deoxyuridylate, a compound which reacts stoichiometrically with a single cysteine. Unlike the other reagents, the inhibition produced by this compound can be completely reversed by added thiols. The same cysteine appears to react with all of the sulfhydryl reagents, as shown by competition experiments and by protection against inactivation by deoxyuridylate. Even at a 100-fold excess of the alkylating agents, only one of the four cysteines in the native enzyme was reactive, attesting to the uniqueness of this residue. Carboxypeptidase A inactivation of the enzyme does not affect either the binding of deoxyuridylate to the enzyme or the reactivity of N-ethylmaleimide with the “catalytic” cysteine. Under denaturing conditions, all four cysteinyl residues react with N-ethylmaleimide or iodoacetate, as shown by identifying the reaction products by amino acid analysis. The covalent ternary complex [(+)5,10-methylenetetrahydrofolate-5-fluorodeoxyuridylate-thymidylate synthetase] (molar ratio = 2:2:1) revealed only two cysteinyl residues capable of reacting with N-ethylmaleimide or iodoacetate upon denaturation. From these data, it appears that one cysteine is involved in the binding of deoxyuridylate and that two of the enzyme's four cysteines are responsible for binding 5-fluorodeoxyuridylate in the ternary complex.     

Thiol-induced reduction of antimony(V) into antimony(III): A comparative study with trypanothione, cysteinyl-glycine, cysteine and glutathione

Gluthathione (GSH) has been previously shown to promote the reduction of pentavalent antimony (Sb(V)) into the more toxic trivalent antimony (Sb(III)) in the antimonial drug, meglumine antimonate. However, this reaction occurred at acidic pH (pH 5) but not at the pH of the cytosol (pH 7.2) in which GSH is encountered. The aim of the present study was to further characterize the reaction between thiols and antimonial drugs, addressing the following aspects: (i) the reducing activity of cysteine (Cys) and cysteinyl-glycine (Cys-Gly), expected to be the predominant thiols in the acidic compartiments of mammalian cells; (ii) the reducing activity of trypanothione (T(SH)₂), the main intracelular thiol in Leishmania parasites; (iii) the influence of the state of complexation of Sb(V) on the rate of Sb(V) reduction. We report here that Cys, Cys-Gly and T(SH)₂ did promote the reduction of Sb(V) into Sb(III) at 37 °C. Strikingly, the initial rates of reduction of Sb(V) were much greater in the presence of Cys-Gly, Cys and T(SH)₂ than in the presence of GSH. These reactions occurred at both pH 5 and pH 7 but were favored at acidic pH. Moreover, our data shows that Sb(V) is reduced more slowly in the form of meglumine antimonate than in its non-complexed form, indicating that the complexation of Sb(V) tends to slow down the rate of its reduction. In conclusion, our data supports the hypothesis that Sb(V) is reduced in vivo by T(SH)₂ within Leishmania parasites and by Cys or Cys-Gly within the acidic compartments of mammalian cells.     

The determination of low-molecular-mass thiols with 4-(hydroxymercuric)benzoic acid as a tag using HPLC coupled online with UV/HCOOH-induced cold vapor generation AFS

An alternative analytical method was established for simultaneous determination of main urinary low-molecular-mass (LMM) thiols including cysteine (Cys), cysteinylglycine (Cys–Gly), homocysteine (HCys), γ-glutamyl cysteine (γ-Glu–Cys) and glutathione (GSH) as well as N-acetylcysteine (NAC) using RPLC coupled on line with UV/HCOOH-induced cold vapor generation atomic fluorescence spectrometry (UV/HCOOH–CVG–AFS) with 4-(hydroxymercuric)benzoic acid (PHMB) as a tag. The LMM thiols were stabilized and labeled by PHMB allowing the determination of reduced form thiols (R-thiols) and total thiols (T-thiols) without and with Tris-(2-carboxyethyl)-phosphine reduction. UV/HCOOH-induced Hg cold vapor generation was used instead of K₂SO₈–KBH₄/NaOH–HCl and/or KBrO₃/KBr–KBH₄/NaOH–HCl systems as an effective interface between RPLC and CVG–AFS. The limits of detection (3σ) of RPLC–(UV/HCOOH)–CVG–AFS with PHMB labeling for Cys, HCys, Cys–Gly, γ-Glu–Cys and GSH as well as NAC were 4.6, 5.9, 5.9, 8.1, 7.3 and 5.9 nM with the RSD of 4.4, 5.1, 3.6, 7.5 4.2 and 3.7% (n = 6 at 2 μM), respectively, satisfying the simultaneous determination of the main urinary LMM thiols. This developed method was applied successfully to determine the LMM R-thiols and T-thiols in 10 urine samples contributed by 10 healthy volunteers.     

DETERMINATION OF CELLULAR LEVELS OF NONPROTEIN THIOLS IN PHYTOPLANKTON AND THEIR CORRELATIONS WITH SUSCEPTIBILITY TO MERCURY1

We determined the intracellular contents and concentrations of cysteine and glutathione in five species of marine phytoplankton, Tetraselmis tetrathele (West) Butcher (Prasinophyceae), Porphyridium purpureum (Bory) Drew et Ross (Rhodophyceae), Pavlova sp. (Haptophyceae), Isochrysis sp. (Haptophyceae), and Pleurochrysis carterae (Braarud et Fagerl) Christensen (Haptophyceae), and examined relationships to mercury susceptibility. Intracellular contents (concentrations) of nonprotein thiols in the five species ranged from 119 to 1210 amol (0.66–12.0 mM) for cysteine, 78 to 719 amol (0.65–2.52 mM) for cystine, 31 to 677 amol (0.13–1.25 mM) for reduced glutathione (GSH), and 12 to 123 amol (0.15–0.26 mM) for oxidized glutathione (GSSG). The intracellular contents of the nonprotein thiols were not proportional to the intracellular concentrations because the cell sizes differed. Oxidation ratios of cysteine:cystine and GSH:GSSG were also wide ranging in the five species, and the higher the concentration of the reduced form of nonprotein thiols, the less they tended to be oxidized. Flow cytometric analyses with fluorescein diacetate were used to monitor the effect of HgCl₂ on esterase, and the 50% effect concentrations (EC₅₀) were compared in the five species. The EC₅₀ after 3 h exposure to HgCl₂ correlated well with the GSH concentrations but not with those of cysteine. These results indicate that the intracellular concentrations of the nonprotein thiols reflect antioxidant activity and susceptibility to heavy metals.     

Thiol metabolism play significant role during cadmium detoxification by Ceratophyllum demersum L.

In the present study, the level of thiols and activity of related enzymes were investigated in coontail (Ceratophyllum demersum L.) plants to analyze their role in combating the stress caused upon exposure to cadmium (Cd; 0–10 μM) for a duration up to 7 d. Plants showed the maximum accumulation of 1293 μg Cd g^?1 dw after 7 d at 10 μM. Significant increases in the level of total non-protein thiols (NP-SH) including phytochelatins (PCs) as well as upstream metabolites of the PC biosynthetic pathway, cysteine and glutathione (GSH) were observed. In addition, significant increases in the activities of cysteine synthase (CS), glutathione-S-transferase (GST), glutathione reductase (GR), as well as in vitro activation of phytochelatin synthase (PCS), were noticed in response to Cd. In conclusion, under Cd stress, plants adapted to a new metabolic equilibrium of thiols through coordinated synthesis and consumption to combat Cd toxicity and to accumulate it.     

Leishmania donovani vs immunity: T-cells sensitized from Leishmania of one donor may modulate their cytokines pattern on re-stimulation with Leishmania from different donor in visceral leishmaniasis

Antimony resistance is frequently encountered during treatment of visceral leishmaniasis (VL) and the differences are well characterized by inadequate IFN-γ dominant type-1 protection mechanisms. The part played by Leishmania parasites derived from antimony treated patients in the outcome of an immune response largely remains to be investigated. In the present study we observed that macrophages of BALB/c mice infected with antimony non-responder (SAG-NR) isolates had a greater amastigote burden than antimony responder (SAG-R) isolates. Later it was observed that antigen from SAG-NR and R L. donovani isolates elicit different cytokine responses in peritoneal blood mononuclear cells (PBMCs) from patients with VL. The production of IFN-γ by T-cells in VL patients increased in response to Leishmania derived from responder patients but this response within same T-cells was lower when sensitized from Leishmania from a non-responder VL patient. On the other hand, IL-4 and IL-10 expression was increased when primed with parasites from non-responder VL source. Such a differential pattern of cytokine expression by the same T-cell population produced to Leishmania from different donors, needs further exploration.     

Species-Specific Antimonial Sensitivity in Leishmania Is Driven by Post-Transcriptional Regulation of AQP1

Leishmania is a digenetic protozoan parasite causing leishmaniasis in humans. The different clinical forms of leishmaniasis are caused by more than twenty species of Leishmania that are transmitted by nearly thirty species of phlebotomine sand flies. Pentavalent antimonials (such as Pentostam or Glucantime) are the first line drugs for treating leishmaniasis. Recent studies suggest that pentavalent antimony (Sb(V)) acts as a pro-drug, which is converted to the more active trivalent form (Sb(III)). However, sensitivity to trivalent antimony varies among different Leishmania species. In general, Leishmania species causing cutaneous leishmaniasis (CL) are more sensitive to Sb(III) than the species responsible for visceral leishmaniasis (VL). Leishmania aquaglyceroporin (AQP1) facilitates the adventitious passage of antimonite down a concentration gradient. In this study, we show that Leishmania species causing CL accumulate more antimonite, and therefore exhibit higher sensitivity to antimonials, than the species responsible for VL. This species-specific differential sensitivity to antimonite is directly proportional to the expression levels of AQP1 mRNA. We show that the stability of AQP1 mRNA in different Leishmania species is regulated by their respective 3’-untranslated regions. The differential regulation of AQP1 mRNA explains the distinct antimonial sensitivity of each species.     

Influence of zinc on cadmium-induced toxicity in nodules of pigeonpea (Cajanus cajan L. Millsp.) inoculated with arbuscular mycorrhizal (AM) fungi

Arbuscular mycorrhizal (AM) fungi are known to alleviate heavy-metal stress in plants. The intent of the present work was to analyze accumulation of heavy metals (Cd and Zn) in nodules of two Cajanus cajan (L.) Millsp. genotypes and their subsequent impact on nitrogen fixation, oxidative stress, and non-protein thiols (glutathione and phytochelatins) with and without AM fungus Glomus mosseae. Accumulation of Cd and Zn in nodules resulted in sharp reduction in nodule number, nodule dry mass as well as nitrogen fixation (leghemoglobin and nitrogenase (N₂ase)), although Cd had more pronounced effects than Zn. Cd-induced lipid peroxidation, H₂O₂ accumulation, and electrolyte leakage were largely reversed by Zn supplementation. Zn application significantly altered the negative effects of Cd on the synthesis of non-protein thiols, suggesting antagonistic behaviour of Zn. Higher concentration of Zn was more effective in lessening the negative effects of Cd than its lower concentration. Remarkable genotypic variation was found, with more severe effects of both the metals in P792 than Sel 85N. Glomus mosseae attenuated the phytotoxic effects of metals in nodules by decreasing metal uptake, oxidative stress, and by enhancing defense system ultimately leading to better nitrogen-fixing potential of pigeonpea nodules.     

Reaction of vanadium(V) with thiols generates vanadium (IV) and thiyl radicals

The in vivo toxicity of vanadium(V) has been found to correlate with the depletion of cellular glutathione and related non-protein thiols. With a view to understanding the mechanism for this observation, we have investigated the oxidation of glutathione, cysteine N-acetylcysteine and penicillamine by vanadium(V), using electron spin resonance (ESR) and ESR spin trapping methodology. The spin trap used was 5,5-dimethyl-1-pyrroline 1-oxide (DMPO). It is found that the oxidation of these thiols by vanadium(V) generates the corresponding thiyl radicals and vanadium- (IV) complexes. The results suggest that free radical reactions play a significant role in the depletion of cellular thiols by vanadium(V) and hence in vanadium(V) toxicity.     

The reaction of carbonyl cyanide phenylhydrazones with thiols

Carbonyl cyanide phenylhydrazone and its ring-substituted analogs react with thiols (thioglycolic acid, 2-mercaptoethanol, dithiothreitol) and amino-thiols (cysteine, glutathione) to give corresponding N-(substituted phynyl)-N′-(alkythiodicyano)-methylhydrazine derivatives. These addition products decompose to the original components in alkaline solution. On the other hand, in the presence of an excess of thiols in aqueous buffered systems the addition reactions are practically quantitative with respect to phenylhydrazones, follow pseudo-first-order kinetics and can be investigated spectrophotometrically. These reactions are of the bimolecular Ad_N type where the non-dissociated form of carbonyl cyanide phenylhydrazones function as an electrophilic component, while the RS^? ion plays the role of nucleophilic component in the case of thiols (the attack of the azomethine group).The reactivity of carbonyl cyanide phenylhydrazones with respect to thiols increases in the order carbonyl cyanide phenylhydrazone < carbonyl cyanide m-chlorophenylhydrazone < carbonyl cyanide p-trifluoromethoxyphenylhydrazone which corresponds to the order of decreasing values of the pK_a constants. On the other hand, the reactivity of thiols increases with their basicity.The reactivity of carbonyl cyanide phynylhydrazone with thiols is comparable with the reactivity of phynyl isothiocyanate and N-ethylmaleimide. It was demostrated that carbonyl cyanide phenylhydrazone is an efficient inhibitor of rabbit muscle glyceraldehyde-3-phosphate dehydrogenase (EC 1.2.1.12).The results obtained are discussed in relation to the biological activity of carbonyl cyanide phenylhydrazones.     

Differential accumulation of cadmium in near-isogenic lines of durum wheat: no role for phytochelatins

Certain cultivars of some crops, including durum wheat (Triticum durum Desf.), have a propensity to accumulate cadmium in the grain. In the 1980s, a Canadian wheat breeding program generated five pairs of near-isogenic lines of durum wheat that vary in cadmium-accumulation. Within each pair, one member accumulates twofold to threefold higher concentrations of cadmium in the shoot and grain. However, the physiological explanation for the high-low phenotype is unknown. We studied correlations between concentrations of cadmium and non-protein thiols, including phytochelatins, in these five pairs of near-isogenic lines to test the hypothesis that differential retention of cadmium-binding complexes in the root would explain the phenotype. The expected high-low pattern of cadmium accumulation was found in three of the pairs. In one pair, cadmium was positively correlated with cysteine and glutathione in the roots and with phytochelatins 2 and 4 in the shoots but in another pair cadmium was strongly negatively correlated with phytochelatins 2 and 4 in the shoots and unrelated to cysteine or glutathione. No correlations between concentrations of cadmium and the non-protein thiols were found in the third pair or in the remaining two pairs. The production of phytochelatins is a well-described response to cadmium but the lack of consistent correlation between cadmium and non-protein thiols in these five near-isogenic lines indicates that complexation with non-protein thiols does not explain differential translocation of cadmium in durum wheat.     

The effect of tumour progression on lymphocyte responses to lectins,measured by fluorescence polarization techniques

The process of fluorochromasia involves the hydrolysis by cells of fluorescein diacetate resulting in an intracellular accumulation of fluorescein. The polarization of the fluorescence of the fluorescein appears to depend on the intracellular fluorescein concentration, the distribution of fluorescein within the cell and the viscosity of the cell cytoplasm.The parameters of fluorochromasia were studied with thymocytes from normal BALB/c mice and from mice bearing an intraperitoneal NK/LY/R lymphoma. During the course of tumour proliferation, the response toT-cell mitogens increased whereas the response to other lectins,e.g. wheat germ agglutinin, decreased or remained unaltered. These changes were consistent with the corresponding increase in immunocompetent cells within the thymus, observed by microelectrophoresis. Thus this sensitive technique provides a useful quantitative assessment of the lectin-lymphoid cell interaction.     

Flow cytometry as a method for assaying the biological activity of phytotoxins

A flow cytometric method has been developed for the qualitative and quantitative evaluation of the biological activities of phytotoxins from plant pathogenic fungi. The method utilized fresh wheat (Triticum aestivum L.) leaf protoplast preparations treated with purified phytotoxins, triticone A-B and triticone D. Subsequently, protoplasts were exposed to fluorescein diacetate, and analyzed by flow cytometry. Information acquired included fluorescence owing to esterase activity on fluorescein diacetate, and chlorophyll autofluorescence. Results indicate that triticone A-B has a rapid dose-dependent toxic effect on wheat protoplasts but triticone D has no toxic effect. This method can also yield information on the mechanism of action of phytotoxins that are relatively unstable or available only in small quantities.     

Regulation of Assimilatory Sulfate Reduction by Herbicide Safeners in Zea mays L

Effects of the herbicide safeners N,N-diallyl-2,2-dichloroacetamide and 4-dichloroacetyl-3,4-dihydro-3-methyl-2H-1,4-benzooxazin (CGA 154281) on the contents in cysteine and glutathione, on the assimilation of ³⁵SO₄²⁻, and on the enzymes of assimilatory sulfate reduction were analyzed in roots and primary leaves of maize (Zea mays) seedlings. Both safeners induced an increase in cysteine and glutathione. In labeling experiments using ³⁵SO₄²⁻, roots of plants cultivated in the presence of safeners contained an increased level of radioactivity in glutathione and cysteine as compared with controls. A significant increase in uptake of sulfate was only detected in the presence of CGA 154281. One millimolar N,N-diallyl-2,2-dichloroacetamide applied to the roots for 6 days increased the activity of adenosine 5′-phosphosulfate sulfotransferase about 20- and threefold in the roots and leaves, respectively, compared with controls. CGA 154281 at 10 micromolar caused a sevenfold increase of this enzyme activity in the roots, but did not affect it significantly in the leaves. A significant increase in ATP-sulfurylase (EC 2.7.7.4) activity was only detected in the roots cultivated in the presence of 10 micromolar CGA 154281. Both safeners had no effect on the activity of sulfite reductase (EC 1.8.7.1) and O-acetyl-l-serine sulfhydrylase (EC 4.2.99.8). The herbicide metolachlor alone or combined with the safeners induced levels of adenosine 5′-phosphosulfate sulfotransferase, which were higher than those of the appropriate controls. Taken together these results show that the herbicide safeners increased both the level of adenosine 5′-phosphosulfate sulfotransferase activity and of the thiols cysteine and glutathione. This indicates that these safeners may be involved in eliminating the previously proposed regulatory mechanism, in which increased concentrations of thiols regulate assimilatory sulfate reduction by decreasing the activities of the enzymes involved.     

Necrotic and apoptotic cell death induced by Captan on <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis>

Captan is one of the most widely used broad-spectrum fungicide applied to control several early and late diseases of grapes, apples, and other fruits and vegetables, and as other phthalimide fungicides is defined as a multisite compound with thiol-reactivity. Captan can affect non-target organisms as yeasts, modifying microbial populations and fermentation processes. In this study, we asked whether Captan thiol-reactivity and other mechanisms are involved in acute Captan-induced cell death on aerobic growing Saccharomyces cerevisiae. Thus for, we analyze cellular protein and non-protein thiols, cell membrane integrity, reactive oxygen species accumulation, phosphatidylserine externalization, and apoptotic mutants behavior. The results showed that when submitted to acute Captan treatment most cells lost their membrane integrity and died by necrosis due to Captan reaction with thiols. However, part of the cells, even maintaining their membrane integrity, lost their culture ability. These cells showed an apoptotic behavior that may be the result of non-protein thiol depletion and consequent increase of reactive oxygen species (ROS). ROS accumulation triggers a metacaspase-dependent apoptotic cascade, as shown by the higher viability of the yca1-deleted mutant. Together, necrosis and apoptosis are responsible for the high mortality detected after acute Captan treatment of aerobically growing cells of S. cerevisiae.     

S-carbamoylation impairs the oxidant scavenging activity of cysteine: its possible impact on increased LDL modification in uraemia

Carbamoylation is the non-enzymatic reaction of cyanate with amino-, hydroxy- or thiol groups. In vivo, amino group modification (N-carbamoylation) resulting in altered function of proteins/amino acids has been observed in patients suffering from uraemia due to urea-derived cyanate. Uraemia has been linked to impaired antioxidant defense. As thiol-compounds like cysteine, N-acetyl cysteine and GSH have oxidant scavenging properties one may speculate that thiol-group carbamoylation (S-carbamoylation) may impair their protective activity. Here we report on the effect of S-carbamoylation on the ABTS free radical and HOCl scavenging property of cysteine as well on its ability to protect LDL from atherogenic modification induced by AAPH generated peroxylradicals or HOCl. The results show that S-carbamoylation impaired the ABTS free radical and HOCl scavenging property of the thiol-compounds tested. The ability of the thiols to protect LDL from lipid oxidation and apolipoprotein modification was strongly diminished by S-carbamoylation. The data indicate that S-carbamoylation could impair the free radical and HOCl scavenging of thiol-amino acids reducing their protective property against LDL atherogenic modification by these oxidant species. As S-carbamoylation is most effective at pH 7 to 5 in vivo thiol-carbamoylation may especially occur at sites of acidic extracellular pH as in hypoxic/inflammatory macrophage rich areas like the atherosclerotic plaque where increased LDL oxidation has been found and may contribute to the higher oxidative stress in uraemia.     

Determination of intracellular glutathione in human skeletal muscle by reversed-phase high-performance liquid chromatography

A chromatographic method for the specific determination of cellular low molecular mass thiols has been applied to human muscle tissue. The method is based on the derivatisation of thiols using monobromobimane, which is a specific reagent for the sulphydryl group. The glutathione and cysteine bimane adducts were separated by reversed-phase HPLC, whilst quantitation of the cysteine and glutathione adducts was achieved by fluorescence spectroscopy. The method was found to yield a quantitative recovery of glutathione (ca. 96%), to be sensitive (down to 20 pmol glutathione/per injection) and reveal a low intra-individual coefficient of variation (C.V. < 5%) of the glutathione concentrations in human skeletal muscle. The concentrations of reduced and total glutathione were 1320 ± 37 μmol/kg wet weight (mean ± S.E.M.) and 1525 ± 66 μmol/kg wet weight, respectively. The method was also applied to tissues from nine healthy volunteers to determine if fluctuations in glutathione level occurred over a 24-h period. No diurnal variation of glutathione level in human skeletal muscle was observed.     

Antimicrobial Peptide-induced Apoptotic Death of Leishmania Results from Calcium-de pend ent, Caspase-independent Mitochondrial Toxicity

α- and θ-defensin-, magainin-, and cathelicidin-type antimicrobial peptides (AMPs) can kill the pathogenic protozoan Leishmania. Comparative studies of a panel of AMPs have defined two distinct groups: those that induce nonapoptotic (Class I) and apoptotic (Class II) parasite killing based on their differential ability to induce phosphatidyl serine exposure, loss of mitochondrial membrane potential and decreased ATP production, induction of caspase-3/7 and -12 activity, and DNA degradation. Class II AMPs cause rapid influx of the vital stain SYTOX and an increase in intracellular Ca²⁺, whereas Class I AMPs cause a slow accumulation of SYTOX and do not affect intracellular Ca²⁺ levels. Inhibitors of cysteine or caspase proteases diminished fast influx of SYTOX through the surface membrane and DNA degradation but do not ablate the annexin V staining or the induction of apoptosis by Class II AMPs. This suggests that the changes in surface permeability in AMP-mediated apoptosis are related to the downstream events of intracellular cysteine/caspase activation or the loss of ATP. The activation of caspase-12-like activity was Ca²⁺-dependent, and inhibitors of voltage-gated and nonspecific Ca²⁺ channels diminished this activity. Flufenamic acid, a nonspecific Ca²⁺ inhibitor, completely ablated AMP-induced mitochondrial dysfunction and cell death, indicating the importance of dysregulation of Ca²⁺ in antimicrobial peptide-induced apoptosis.Leishmania are vector-borne protozoa that parasitize host macrophage phagolysosomes. Activated macrophages kill intracellular parasites in part by induction of apoptosis by nitric oxide in a caspase- and cysteine protease-independent manner (1, 2). Limited apoptosis of Leishmania within the sandfly vector, prior to infection of host, may be important to the establishment of infection (3, 4). In vitro Leishmania can undergo apoptosis by exposure to many different compounds that lead to alteration of surface the cell membrane causing enhanced annexin V staining, activation of caspase-like proteases, DNA laddering, and mitochondrial membrane depolarization. Our work has recently shown that antimicrobial peptide exposure of Leishmania can induce apoptosis, which occurs concomitant with activation of both caspase and cysteine protease activities (5).Antimicrobial peptides (AMPs)2 are structurally diverse highly cationic proteins between 10 and 50 amino acids and are components of the innate immune systems of organisms within all kingdoms. AMPs have a myriad of functions and are known to interact with and disrupt microbial surface membranes leading to cell death. Magainins are α-helical AMPs expressed in the skin of frogs, whereas cathelicidins are mammalian β-pleated sheet peptides, both of which can kill Leishmania at physiological concentrations (5). We have documented that magainins are quite apt at causing AMP-induced apoptosis of surface metalloprotease null mutants of L. major and that these exhibited features of apoptotic Leishmania induced by different stimuli (5). These two different structurally diverse classes of AMPs are known to interact with membranes in distinct ways (6), yet it is unknown whether we they can kill parasites via disparate mechanisms.Apoptosis of Leishmania induced by antimony, camptothecin, and hydrogen peroxide all induce significant increases in cytosolic calcium, which is toxic to mitochondrial membrane potential (7–9). Calcium-related cell death and mitochondrial toxicity are related to the activity of nonspecific calcium channels (7, 8, 10), which may be attributable to their location in the mitochondrial membrane (11, 12). Apoptosis of Leishmania is also associated with increases in caspase-like and cysteine protease activity (5, 7, 9, 13, 14). Caspase-3/7-like activation can be induced by a number of stimuli despite the absence of genes encoding caspases within the L. major genome (15). In addition, the ancestral metacaspase expressed by Leishmania donovani is a trypsin-like protease not inhibitible with caspase inhibitors (16). We and others have found that cysteine protease activity may be important for certain features of Leishmania apoptosis such as DNA degradation (5). It is quite clear that Leishmania can undergo apoptosis in caspase-dependent and caspase-independent pathways (17), yet we do not know what mode of apoptosis occurs upon AMP exposure.In the work presented here we show that two structurally diverse AMPs kill Leishmania differentially inducing either nonapoptotic or apoptotic cell death. We have characterized AMP-induced apoptotic cell death and found that although it is associated with the activation of caspase-3/7- and -12-like activities, these activities are not essential to apoptosis. Conversely we find that a dramatic increase in cytosolic Ca²⁺ correlates with the mitochondrial membrane dysfunction and the decline in ATP production and cell death and that this is completely inhibited by blockade of nonspecific calcium channels. Blockade of caspase and cysteine proteases affect surface membrane permeability changes and DNA degradation but do not prevent cell death, suggesting that AMP-induced apoptosis may be a caspase-independent process. These data provide a framework to understand the critical events in AMP-mediated apoptosis and may be helpful in the rational design of chemotherapeutic strategies for manipulation of cell death pathways in Leishmania.