Nitric oxide-induced eosinophil apoptosis is dependent on mitochondrial permeability transition (mPT), JNK and oxidative stress: apoptosis is preceded but not mediated by early mPT-dependent JNK activation

Background

Eosinophils are critically involved in the pathogenesis of asthma. Nitric oxide (NO) is produced in high amounts in asthmatic lungs and has an important role as a regulator of lung inflammation. NO was previously shown to induce eosinophil apoptosis mediated via c-jun N-terminal kinase (JNK) and caspases. Our aim was to clarify the cascade of events leading to NO-induced apoptosis in granulocyte macrophage-colony stimulating factor (GM-CSF)-treated human eosinophils concentrating on the role of mitochondria, reactive oxygen species (ROS) and JNK.

Methods

Apoptosis was determined by flow cytometric analysis of relative DNA content, by Annexin-V labelling and/or morphological analysis. Immunoblotting was used to study phospho-JNK (pJNK) expression. Mitochondrial membrane potential was assessed by JC-1-staining and mitochondrial permeability transition (mPT) by loading cells with calcein acetoxymethyl ester (AM) and CoCl₂ after which flow cytometric analysis was conducted. Statistical significance was calculated by repeated measures analysis of variance (ANOVA) or paired t-test.

Results

NO-donor S-nitroso-N-acetyl-D,L-penicillamine (SNAP) induced late apoptosis in GM-CSF-treated eosinophils. SNAP-induced apoptosis was suppressed by inhibitor of mPT bongkrekic acid (BA), inhibitor of JNK SP600125 and superoxide dismutase-mimetic AEOL 10150. Treatment with SNAP led to late loss of mitochondrial membrane potential. Additionally, we found that SNAP induces early partial mPT (1 h) that was followed by a strong increase in pJNK levels (2 h). Both events were prevented by BA. However, these events were not related to apoptosis because SNAP-induced apoptosis was prevented as efficiently when BA was added 16 h after SNAP. In addition to the early and strong rise, pJNK levels were less prominently increased at 20–30 h.

Conclusions

Here we demonstrated that NO-induced eosinophil apoptosis is mediated via ROS, JNK and late mPT. Additionally, our results suggest that NO induces early transient mPT (flickerings) that leads to JNK activation but is not significant for apoptosis. Thereby, we showed some interesting early events in NO-stimulated eosinophils that may take place even if the threshold for irreversible mPT and apoptosis is not crossed. This study also revealed a previously unknown physiological function for transient mPT by showing that it may function as initiator of non-apoptotic JNK signalling.     

Catalpol protects primary cultured cortical neurons induced by Aβ1–42 through a mitochondrial-dependent caspase pathway

It has been reported that catalpol, an iridoid glucoside, isolated from the root of Rehmannia glutinosa, protected cells from damage induced by a variety of toxic stimulus such as LPS, MPP⁺ and rotenone. Here, we further evaluated the effect of catalpol against Aβ_1–42-induced apoptosis in primary cortical neuron cultures. In the present study, the primary cortical neuron culture treated with Aβ_1–42 was severed as cell model of Alzheimer's disease (AD) in vitro. By exposure to Aβ_1–42 (5 μM) for 72 h in cultures, neuronal apoptosis occurred characterized by enhancement of activities of caspases and reactive oxygen species (ROS) as well as Bax increase, loss of mitochondrial membrane potential and cytochrome c release. Pretreatment with catalpol (0.5 mM) for 30 min prior to Aβ_1–42 treatment attenuated neuronal apoptosis not only by reversing intracellular ROS accumulation, Bax level, mitochondrial membrane potential and, cytochrome c release to some extent, but also through regulating the activity and cleavage of caspase-3 and caspase-9. Thus, catalpol protects primary cultured cortical neurons induced by Aβ_1–42 through a mitochondrial-dependent caspase pathway.     

The biological functions of polyamine oxidation products by amine oxidases: Perspectives of clinical applications

Summary. The polyamines spermine, spermidine and putrescine are ubiquitous cell components. If they accumulate excessively within the cells, due either to very high extracellular concentrations or to deregulation of the systems which control polyamine homeostasis, they can induce toxic effects. These molecules are substrates of a class of enzymes that includes monoamine oxidases, diamine oxidases, polyamine oxidases and copper containing amine oxidases. Polyamine concentrations are high in growing tissues such as tumors. Amine oxidases are important because they contribute to regulate levels of mono- and polyamines. These enzymes catalyze the oxidative deamination of biogenic amines and polyamines to generate the reaction products H₂O₂ and aldehyde(s) that are able to induce cell death in several cultured human tumor cell lines. H₂O₂ generated by the oxidation reaction is able to cross the inner membrane of mitochondria and directly interact with endogenous molecules and structures, inducing an intense oxidative stress. Since amine oxidases are involved in many crucial physiopathological processes, investigations on their involvement in human diseases offer great opportunities to enter novel classes of therapeutic agents.     

Thiolation and nitrosation of cysteines in biological fluids and cells

Summary. Thiols (RSH) are potent nucleophilic agents, the rates of which depend on the pKa of the sulfhydryl. Unlike compounds having other nucleophile moieties (–OH or –NH₂), RSH are involved in reactions, such as conjugations, redox and exchange reactions. Although protein SH groups (PSH) react like non-protein thiols (NPSH), the biochemistry of proteins is much more complex for reasons such as steric hindrance, charge distribution and accessibility of PSH to the solvent (protein conformation). The reaction rates and types of end-products of PSH vary a lot from protein to protein. The biological problem is even more complex because in all compartments and tissues, there may be specific competition between thiols (namely between GSH and PSH), regulated by the properties of antioxidant enzymes. Moreover, PSH are divided biologically into essential and non-essential and their respective influence in the various biological systems is unknown. It follows that during phenomena eliciting a prompt thiol response (oxidative stress), the antioxidant PSH response and reaction mechanisms vary considerably from case to case. For example, in spite of a relatively low pKa that should guarantee good antioxidant capacity, PSH of albumin has much less propensity to form adducts with conjugating agents than NPSH; moreover, the structural characteristics of the protein prevent albumin from forming protein disulfides when exposed to oxidants (whereas protein-thiol mixed disulfides are formed in relative abundance). On the other hand, proteins with a relatively high reactivity, such rat hemoglobin, have much greater antioxidant capacity than GSH, but although human hemoglobin has a pKa similar to GSH, for structural reasons it has less antioxidant capacity than GSH.When essential PSH are involved in S-thiolation and S-nitrosation reactions, a similar change in biological activity is observed. S-thiolated proteins are a recurrent phenomenon in oxidative stress elicited by reactive oxygen species (ROS). This event may be mediated by disulfides, that exchange with PSH, or by the protein intermediate sulfenic acid that reacts with thiols to form protein-mixed disulfides. During nitrosative stress elicited by reactive nitrogen species (RNS), depending on the oxygen concentration of the system, nitrosation reactions of thiols may also be accompanied by protein S-thiolation. In this review we discuss a number of cell processes and biochemical modifications of enzymes that indicate that S-thiolation and S-nitrosation may occur simultaneously in the same protein in the presence of appropriate interactions between ROS and RNS.     

Synthesis of modular dipeptide mimetics on the basis of diazabicycloalkanes and derivatives thereof with sulphur containing side chains

Summary. We present the synthesis of new modular dipeptide mimetics based on diazabicycloalkane backbones. These diazabicycloalkanes are ligands for the prostate specific membrane antigen (PSMA), a well known tumor marker. Our previously described synthetic route to enantiomerically pure diazabicycloalkanes is extended to yield polyfunctional diazabicycloalkanes with a modular character using a new ring closing methodology. This, finally, allows us to attach linker moieties to different positions of the diazabicycloalkane scaffold providing conjugation sites to other functional molecules such as markers or cytostatic compounds. Furthermore, successful synthesis of sulphur-containing dipeptide analogues as for example CysX_AA- or HCysX_AA-mimetics on the basis of diazabicycloalkanes is described.     

Oxidative stress and mitochondrial dysfunction play a role in myelodysplastic syndrome development,diagnosis, and prognosis: A pilot study

Abstract

The imbalance between reactive oxygen species (ROS) production and their elimination by antioxidants leads to oxidative stress. Depending on their concentration, ROS can trigger apoptosis or stimulate cell proliferation. We hypothesized that oxidative stress and mitochondrial dysfunction may participate not only in apoptosis detected in some myelodysplastic syndrome (MDS) patients, but also in increasing proliferation in other patients. We investigated the involvement of oxidative stress and mitochondrial dysfunction in MDS pathogenesis, as well as assessed their diagnostic and prognostic values. Intracellular peroxides, superoxide, superoxide/peroxides ratio, reduced glutathione (GSH), and mitochondrial membrane potential (Δψ_mit) levels were analyzed in bone marrow cells from 27 MDS patients and 12 controls, by flow cytometry. We observed that all bone marrow cell types from MDS patients had increased intracellular peroxide levels and decreased GSH content, compared with control cells. Moreover, oxidative stress levels were MDS subtype— and risk group—dependent. Low-risk patients had the highest ROS levels, which can be related with their high apoptosis; and intermediate-2-risk patients had high Δψ_mit that may be associated with their proliferative potential. GSH levels were negatively correlated with transfusion dependency, and peroxide levels were positively correlated with serum ferritin level. GSH content proved to be an accurate parameter to discriminate patients from controls. Finally, patients with high ROS or low GSH levels, as well as high superoxide/peroxides ratio had lower overall survival. Our results suggest that oxidative stress and mitochondrial dysfunction are involved in MDS development, and that oxidative stress parameters may constitute novel diagnosis and/or prognosis biomarkers for MDS.     

Mitochondrial reactive oxygen species in cell death signaling

During apoptosis, mitochondrial membrane permeability (MMP) increases and the release into the cytosol of pro-apoptotic factors (procaspases, caspase activators and caspase-independent factors such as apoptosis-inducing factor (AIF)) leads to the apoptotic phenotype. Apart from this pivotal role of mitochondria during the execution phase of apoptosis (documented in other reviews of this issue), it appears that reactive oxygen species (ROS) produced by the mitochondria can be involved in cell death. These toxic compounds are normally detoxified by the cells, failing which oxidative stress occurs. However, ROS are not only dangerous molecules for the cell, but they also display a physiological role, as mediators in signal transduction pathways. ROS participate in early and late steps of the regulation of apoptosis, according to different possible molecular mechanisms. In agreement with this role of ROS in apoptosis signaling, inhibition of apoptosis by anti-apoptotic Bcl-2 and Bcl-x(L) is associated with a protection against ROS and/or a shift of the cellular redox potential to a more reduced state. Furthermore, the fact that active forms of cell death in yeast and plants also involve ROS suggests the existence of an ancestral redox-sensitive death signaling pathway that has been independent of caspases and Bcl-2.     

Betulinic Acid Induces Apoptosis in Differentiated PC12 Cells Via ROS-Mediated Mitochondrial Pathway

Betulinic acid (BA), a pentacyclic triterpene of natural origin, has been demonstrated to have varied biologic activities including anti-viral, anti-inflammatory, and anti-malarial effects; it has also been found to induce apoptosis in many types of cancer. However, little is known about the effect of BA on normal cells. In this study, the effects of BA on normal neuronal cell apoptosis and the mechanisms involved were studied using differentiated PC12 cells as a model. Treatment with 50 μM BA for 24 h apparently induced PC12 cell apoptosis. In the early stage of apoptosis, the level of intracellular reactive oxygen species (ROS) increased. Afterwards, the loss of the mitochondrial membrane potential, the release of cytochrome c and the activation of caspase-3 occurred. Treatment with antioxidants could significantly reduce BA-induced PC12 cell apoptosis. In conclusion, we report for the first time that BA induced the mitochondrial apoptotic pathway in differentiated PC12 cells through ROS.     

Monoamine oxidase‐A is a novel driver of stress‐induced premature senescence through inhibition of parkin‐mediated mitophagy

Cellular senescence, the irreversible cell cycle arrest observed in somatic cells, is an important driver of age‐associated diseases. Mitochondria have been implicated in the process of senescence, primarily because they are both sources and targets of reactive oxygen species (ROS). In the heart, oxidative stress contributes to pathological cardiac ageing, but the mechanisms underlying ROS production are still not completely understood. The mitochondrial enzyme monoamine oxidase‐A (MAO‐A) is a relevant source of ROS in the heart through the formation of H₂O₂ derived from the degradation of its main substrates, norepinephrine (NE) and serotonin. However, the potential link between MAO‐A and senescence has not been previously investigated. Using cardiomyoblasts and primary cardiomyocytes, we demonstrate that chronic MAO‐A activation mediated by synthetic (tyramine) and physiological (NE) substrates induces ROS‐dependent DNA damage response, activation of cyclin‐dependent kinase inhibitors p21^cip, p16^ink4a, and p15^ink4b and typical features of senescence such as cell flattening and SA‐β‐gal activity. Moreover, we observe that ROS produced by MAO‐A lead to the accumulation of p53 in the cytosol where it inhibits parkin, an important regulator of mitophagy, resulting in mitochondrial dysfunction. Additionally, we show that the mTOR kinase contributes to mitophagy dysfunction by enhancing p53 cytoplasmic accumulation. Importantly, restoration of mitophagy, either by overexpression of parkin or inhibition of mTOR, prevents mitochondrial dysfunction and induction of senescence. Altogether, our data demonstrate a novel link between MAO‐A and senescence in cardiomyocytes and provides mechanistic insights into the potential role of MAO‐dependent oxidative stress in age‐related pathologies.     

Production of amino acids by Azotobacter vinelandii and Azotobacter chroococcum with phenolic compounds as sole carbon source under diazotrophic and adiazotrophic conditions

Summary. Azotobacter vinelandii strain ATCC 12837 and Azotobacter chroococcum strain H23 (CECT4435) were tested to grow in N-free or NH₄Cl amended chemically defined media, with protocatechuic acid or sodium p-hydroxybenzoate as sole carbon (C) sources at a concentration of 2 mmol/L. Both substrates supported grow at similar rates than bacteria grown in control media amended with 2 mmol/L sodium succinate as C source. The two strains produced aspartic acid, serine, glutamic acid, glycine, hystidine, threonine, arginine, alanine, proline, cysteine, tyrosine, valine, methionine, lysine, isoleucine, leucine and phenylalanine after 72 h of growth in chemically defined media with 2 mmol/L of phenolic compounds or sodium succinate as sole C source amended or unamended with 0.1% (w/v) NH₄Cl. Qualitative and quantitative production of all amino acids was not affected by the use of different C and N substrates.     

β-Carotene-induced apoptosis is mediated with loss of Ku proteins in gastric cancer AGS cells

High dietary intakes and high blood levels of β-carotene are associated with a decreased incidence of various cancers. The anticancer effect of β-carotene is related to its pro-oxidant activity. DNA repair Ku proteins, as a heterodimer of Ku70 and Ku80, play a crucial role in DNA double-strand break repair. Reductions in Ku70/80 contribute to apoptosis. Previously, we showed that reactive oxygen species (ROS) activate caspase-3 which induces degradation of Ku proteins. In the present study, we investigated the mechanism of β-carotene-induced apoptosis of gastric cancer AGS cells by determining cell viability, DNA fragmentation, apoptotic indices (increases in cytochrome c and Bax, decrease in Bcl-2), ROS levels, mitochondrial membrane potential, caspase-3 activity, Ku70/80 levels, and Ku-DNA-binding activity of the cells treated with or without antioxidant N-acetyl cysteine and caspase-3 inhibitor z-DEVED-fmk. As a result, β-carotene induced apoptosis (decrease in cell viability, increases in DNA fragmentation and apoptotic indices) and caspase-3 activation, but decreased Ku70/80 levels and Ku-DNA-binding activity. β-Carotene-induced alterations (increase in caspase-3 activity, decrease in Ku proteins) and apoptosis were inhibited by N-acetyl cysteine and z-DEVED-fmk. Increment of intracellular and mitochondrial ROS levels and loss of mitochondrial membrane potential were suppressed by N-acetyl cysteine, but not by z-DEVED-fmk in β-carotene-treated cells. Therefore, β-carotene-induced increases in ROS and caspase-3 activity may lead to reduction of Ku70/80 levels, which results in apoptosis in gastric cancer cells. Loss of Ku proteins might be the underlying mechanism for β-carotene-induced apoptosis in gastric cancer cells.     

Induction of yeast apoptosis by an antimicrobial peptide, Papiliocin

Papiliocin is a 37-residue peptide isolated from the swallowtail butterfly Papilio xuthus. In this study, we found that Papiliocin induced the accumulation of reactive oxygen species (ROS) and hydroxyl radicals known to be important regulators of apoptosis in Candida albicans. To examine the relationship between the accumulation of ROS and the induction of apoptosis, we investigated the apoptotic effects of Papiliocin using apoptotic markers. Cells treated with Papiliocin showed a series of cellular changes normally seen in cells undergoing apoptosis: plasma membrane translocation of phosphatidylserine from the inner to the outer membrane leaflet, measured by Annexin V staining, dissipation of the mitochondrial membrane potential, observed by DiOC₆(3) staining; and the presence of active metacaspases, measured using the CaspACE FITC-VAD-FMK, as early apoptotic events. In addition, DNA condensation and fragmentation, which is important marker of late stage apoptosis, was seen by DAPI and TUNEL assay. Therefore, these results suggest that Papiliocin leads to apoptosis in C. albicans via ROS accumulation.     

Reactive oxygen species generation is independent of de novo sphingolipids in apoptotic photosensitized cells

Our recent studies have shown that the de novo sphingolipids play a role in apoptosis of photosensitized cells. To elucidate the involvement of the de novo sphingolipids in reactive oxygen species (ROS) production and mitochondrial depolarization during apoptosis, the stress inducer photodynamic therapy (PDT) with the photosensitizer Pc 4 was used. In Jurkat cells PDT-triggered ROS production or mitochondrial membrane potential (deltapsi(m)) loss was not prevented by the de novo sphingolipid synthesis inhibitor ISP-1. However, PDT + C16-ceramide led to enhanced mitochondrial depolarization and DEVDase activation. The superoxide dismutase mimic manganese (III) tetrakis (4-benzoic acid) porphyrin (MnTBAP) protected Jurkat cells from ROS generation and apoptosis, but not from deltapsi(m) reduction. Sphinganine or C16-ceramide counteracted MnTBAP-induced protection from apoptosis in Jurkat, as well as CHO cells. In LY-B cells, CHO-derived mutants deficient in serine palmitoyltransferase (SPT) activity and the de novo sphingolipid synthesis, mitochondrial depolarization, but not ROS generation, was suppressed post-PDT. In LY-B cells transfected with the SPT component LCB1, deltapsi(m) collapse post-PDT was restored. The data support the following hypotheses: MnTBAP protects against apoptosis via steps downstream of deltapsi(m) loss; de novo sphingolipids are not required for ROS generation, but can play a role in deltapsi(m) dissipation in photosensitized apoptotic cells.     

Bio-available amino acids extraction from soil by demineralized water and 0.5 M ammonium acetate

Summary. The extraction and comparison of soil bio-available amino acids using either demineralised water (DEMI H₂O) or 0.5 M ammonium acetate (0.5 M AAc) solution is reported. Results show that the extraction by 0.5 M AAc is a better method to assess the concentration of bio-available amino acids in soil than DEMI H₂O due to higher extraction efficiency and better amino acid protection against microbial degradation during processing.     

Hyperglycemia induces apoptosis and p53 mobilization to mitochondria in RINm5F cells

The mechanisms related to hyperglycemia-induced pancreatic β-cell apoptosis are poorly defined. Rat insulin-producing cells (RINm5F) cultured in high glucose concentrations (30 mM) showed increased apoptosis and protein p53 translocation to mitochondria. In addition, hyperglycemia induced both the disruption of mitochondrial membrane potential (Δ < eqid1 > _m), and an increase in reactive oxygen species (ROS), as shown by fluorescence changes of JC-1 and dichlorodihydrofluorescein-diacetate (DCDHF-DA), respectively. The increased intracellular ROS by high glucose exposure was blunted by mitochondrial-function and NADPH-oxidase inhibitors. We postulate that the concomitant mobilization of p53 protein to the mitochondria and the subsequent changes on the Δ < eqid2 > _m, lead to an important pancreatic β-cell apoptosis mechanism induced by oxidative stress caused by hyperglycemia. This work is part of the thesis required for the doctorate degree in Biological Sciences at the Universidad Autónoma Metropolitana, Mexico City, Mexico.     

Tailoring Organic Cation of 2D Air‐Stable Organometal Halide Perovskites for Highly Efficient Planar Solar Cells

2D perovskites have recently been shown to exhibit significantly improved environmental stability. Derived from their 3D analogues, 2D perovskites are formed by inserting bulky alkylammonium cations in‐between the anionic layers. However, these insulating organic spacer cations also hinder charge transport. Herein, such a 2D perovskite, (iso‐BA)₂(MA)₃Pb₄I₁₃, that contains short branched‐chain spacer cations (iso‐BA⁺) and shows a remarkable increase of optical absorption and crystallinity in comparison to the conventional linear one, n‐BA⁺, is designed. After applying the hot‐casting (HC) technique, all these properties are further improved. The HC (iso‐BA)₂(MA)₃Pb₄I₁₃ sample exhibits the best ambient stability by maintaining its initial optical absorption after storage of 840 h in an environmental chamber at 20 °C with a relative humidity of 60% without encapsulation. More importantly, the out‐of‐plane crystal orientation of (iso‐BA)₂(MA)₃Pb₄I₁₃ film is notably enhanced, which increases cross‐plane charge mobility. As a result, the highest power conversion efficiencies (PCEs) measured from for current density versus voltage curves afford 8.82% and 10.63% for room‐temperature and HC‐processed 2D perovskites based planar solar cells, respectively. However, the corresponding steady‐state PCEs are remarkably lower, which is presumably due to the significant hysteresis phenomena caused by low charge extraction efficiency at interfaces of C₆₀/2D perovskites.     

Blockade of a Retinal cGMP-gated Channel by Polyamines

The cyclic nucleotide–gated (CNG) channel in retinal rods converts the light-regulated intracellular cGMP concentration to various levels of membrane potential. Blockade of the channel by cations such as Ca²⁺ and Mg²⁺ lowers its effective conductance. Consequently, the membrane potential has very low noise, which enables rods to detect light with extremely high sensitivity. Here, we report that three polyamines (putrescine, spermidine, and spermine), which exist in both the intracellular and extracellular media, also effectively block the CNG channel from both sides of the membrane. Among them, spermine has the greatest potency. Extracellular spermine blocks the channel as a permeant blocker, whereas intracellular spermine appears to block the channel in two conformations—one permeant, and the other non- (or much less) permeant. The membrane potential in rods is typically depolarized to approximately −40 mV in the dark. At this voltage, K _1/2 of the CNG channel for extracellular spermine is 3 μM, which is 100–1,000-fold higher affinity than that of the NMDA receptor-channel for extracellular spermine. Blockade of the CNG channel by polyamines may play an important role in suppressing noise in the signal transduction system in rods.     

The influence of calcium and reactive oxygen species on influenza virus-induced apoptosis

In previous studies we observed that influenza A and B viruses induce apoptosis in Madin-Darby canine kidney (MDCK) cells and that this apoptosis is blocked by expression of bcl-2. Using a well-characterized, highly virulent, avian influenza virus, A/Turkey/Ontario/7732/66 (H5N9) (Ty/Ont), we sought to better understand this system. We investigated the influence of two cellular factors that are known to function in other models of apoptosis inhibited by bcl-2, calcium (Ca(2+)) and reactive oxygen species (ROS). Although Ca(2+) chelators generally inhibit apoptosis, treatment of MDCK cells with either an extracellular chelator, EGTA, or an intracellular chelator, BAPTAAM, induced apoptosis instead and enhanced Ty/Ont-induced apoptosis. Conversely, treatment with an ionophore, ionomycin, blocked the viral-induced apoptosis. In terms of ROS, neither treatment with antioxidants, N(2) flushing to induce hypoxia, nor nigericin (a compound which, like bcl-2, stabilizes the mitochondrial membrane potential against the effects of ROS and subsequent Ca(2+) dysregulation) were able to block Ty/Ont-induced apoptosis. Therefore, it is likely that ROS play little, if any, role in influenza-induced apoptosis in MDCK cells and the influence of Ca(2+) appears to be opposite to that in the majority of other more classical models of apoptosis.     

Role of calcium in reactive oxygen species-induced apoptosis in Candida albicans: an antifungal mechanism of antimicrobial peptide,PMAP-23

PMAP-23 (RIIDLLWRVRRPQKPKFVTVWVR-NH₂) is an antimicrobial peptide (AMP) derived from porcine myeloid. Membrane disruption is thought to underpin the anticandidal activity of PMAP-23. However, many AMPs act via mechanisms other than simple membrane permeabilisation. Here, we investigated the anticandidal mechanism of PMAP-23 at low concentrations. Membrane disruption and depolarisation and rapid K⁺ efflux were observed in Candida albicans cells treated with 5?µM PMAP-23. In contrast, 2.5?µM PMAP-23 caused membrane depolarisation and K⁺ efflux without membrane disruption. The lower PMAP-23 concentration increased cytosolic and mitochondrial Ca²⁺ levels. Disruption of Ca²⁺ homeostasis altered the NAD⁺/NADH ratio and resulted in reactive oxygen species (ROS) accumulation and glutathione oxidation. PMAP-23 treatment also stimulated apoptosis, as evidenced by metacaspase activation, DNA fragmentation, and phosphatidylserine externalisation. Pretreatment with the mitochondrial Ca²⁺ uptake inhibitor (ruthenium red) or ROS scavenger (N-acetylcysteine) attenuated these apoptotic events. Our results suggest that PMAP-23 induces apoptosis as antifungal mechanism, and mitochondrial Ca²⁺-induced ROS is major factor to trigger the apoptosis. Thus, the anticandidal activity of PMAP-23 is not based solely on disruption of biological membranes but also involves induction of apoptosis via mitochondrial Ca²⁺-dependent ROS. PMAP-23 mode of action sheds new light on the antifungal mechanism of antimicrobial peptides, supporting the role of Ca²⁺ and ROS in apoptosis regulation.     

Inhibition of the L-dopa transport system in human epidermal Langerhans cells by omeprazole and its analogues

Summary. L-3,4-dihydroxyphenylalanine (L-dopa) transport into human Langerhans cells (LC) occurs by a saturable mediation. This plasma membrane agency is, due to its characteristics, distinguishable from systems transporting other neutral, cationic and anionic amino acids into other cells and serves to catalyze the flow of L-dopa, only, into LC. The uphill operation of this L-dopa transport system is believed to occur by down-gradient countermigration of H⁺. Due to the uniqueness of the L-dopa transport system, the widely used analogue inhibition approach was not applicable. Instead we studied omeprazole and its analogues in our search for suitable inhibitory candidates. Omeprazole and most of its analogues were indeed inhibitory in the concentration range 1–100mol/L. Conspicuously, the compounds with strongest polarity were least inhibitory. The inhibitory pattern displayed by omeprazole and the other analogues on L-dopa uptake in LC corresponded to some extent to what has been observed previously for purified H⁺,K⁺-ATPase from tubulovesicles of the stomach. No effects of the inhibitors were registered on energy charge and lactate production of epidermal biopsies, nor were any gross alterations of ultrastructure of LC noticed.