Interaction of the polyene antibiotic etruscomycin with large unilamellar lipid vesicles: binding and proton permeability inducement

The effect of the polyene antibiotic etruscomycin on the permeability of large unilamellar lipid vesicles was investigated. Proton leakage was induced in egg-yolk phosphatidylcholine (EPC) vesicles only when sterol was present in the membrane; the extent of leakage was limited. High etruscomycin/lipid ratios (R) were necessary (R greater than 0.1). Higher percentages of sterol increased the permeability, slightly more strongly for ergosterol than for cholesterol. Dipalmitoylphosphatidylcholine (DPPC) vesicles were more sensitive to permeability inducement, even in the absence of sterol in the bilayer (inducement for R greater than 0.06). The interactions of etruscomycin with the vesicles were examined by circular dichroism, fluorescence and 31P-NMR. In the range of antibiotic concentration where permeability was induced, R greater than 0.1 for EPC vesicles, R greater than 0.06 for DPPC vesicles, etruscomycin exhibited characteristic circular dichroism spectra independent of the presence of sterol. Under the same conditions, 31P-NMR and fluorescence studies indicated a destruction or a fusion of the vesicle bilayer. At lower etruscomycin concentrations (R less than 0.03), the etruscomycin circular dichroism spectra were different, indicating that the interaction with membranes containing ergosterol differed from that with membranes containing cholesterol. From correlating the increase in fluorescence intensity with this interaction, as well as from exchange experiments, it was inferred that etruscomycin at a low antibiotic/lipid ratio is more strongly bound to ergosterol-containing vesicles than to cholesterol-containing vesicles. These results and their comparison with the results obtained with other polyene antibiotics indicate that at low R etruscomycin resembles amphotericin rather than filipin in its preferential binding to ergosterol-containing vesicles. At higher R, that is in conditions where permeability is induced, the selectivity is different. The corresponding mechanism seems not to involve the formation of an etruscomycin-sterol channel, since the hydrophobic chain of the complex would be too short to form a channel

Antibiotics as tool to investigate cell functional state

In recent years, the use of antibiotics beyond their canonical antibacterial role has come to represent a field with a wide range of applications. Antibiotics have been studied as a tool to overcome drug resistance in cancer cells, modulate enzymatic and inflammatory responses, mark cell function in pathologies related to changes in protein or lipid profile, and regulate cell redox homeostasis in some pathophysiological conditions. For example, myriocin is employed in biochemical research to deplete sphingolipids by inhibiting the de novo synthesis of ceramide, whereas filipin, as a naturally fluorescent polyene antibiotic that selectively binds to cholesterol, is commonly used as an accurate histochemical marker for the diagnosis of disorders that lead to the accumulation of cholesterol in lysosomal organelles. Furthermore, some naturally occurring antibiotics have been studied as a subgroup of small signaling molecules that influence the gene expression of many cellular functions. This Special Issue focuses on the structural and functional aspects of the alternative use of antibiotics and on the continuous progress performed in antibiotic research in the fields of genetics, molecular biology and synthesis by biocatalysis, as well as biotechnological and biomedical applications

Effects of the semisynthetic bis-indole derivative KAR-2 on store-operated calcium entry in human neutrophils

We studied the effect of KAR-2 on cytosolic Ca2+ level in human neutrophils by using a fluorescent dye (Fura-2) trapped in the cells. KAR-2 is a semisynthetic bis-indole derivative that shares vinblastine anti-microtubular properties, but does not share the vinblastine antagonistic effect on calmodulin. Therefore KAR-2 offers a convenient mean of studying the effect of microtubule destabilization, without concomitant calmodulin alterations. We found that KAR-2 induces Ca2+ release from intracellular stores, whereby the stores are depleted. In addition KAR-2 reduces store-operated entry of extracellular Ca2+ induced by agonists such as thapsigargin or ATP. On the other hand, in Ca2+ refilled cells, KAR-2 promotes limited entry of extracellular Ca2+ in the absence of agonist, but still interferes prominently with Ca2+ entry triggered by ATP and with Ca2+ uptake by intracellular stores. We suggest that Ca2+ traffic through the plasma membrane is operated by two diverse pathways: the prominent pathway is interfered with by microtubule destabilization, while an alternate and minor pathway is actually favored (or uncovered) following microtubule destabilization

POLYENE ANTIBIOTICS INHIBIT SUPEROXIDE-PRODUCING NADPH OXIDASE IN A POLYMORPHONUCLEAR CELL-FREE SYSTEM

We studied the effect of polyene macrolide antibiotics on the NADPH-dependent superoxide production induced by arachidonic acid in a cell-free system consisting of the membrane and cytosolic fractions obtained from bovine polymorphonuclear leukocytes. Preincubation of the membrane fraction with polyenes before addition of the soluble components of the reaction mixture resulted in a dose-dependent inhibition of superoxide production

Thiotaurine modulates human neutrophil activation

Neutrophils are well recognized as one of the major players during acute infl ammation.They are typically the fi rst leukocytes to be recruited to an infl ammatory site and can eliminate pathogens by multiple means. Two different microbicidal mechanisms occur within the neutrophils: the oxidative and the non oxidative systems. The oxygen-dependent mechanism acts through generation of reactive oxygen species (ROS), and the oxygen-independent mechanism acts through production of antimicrobial peptides and proteolytic enzymes. During infl ammation, neutrophils are activated in response to several agonists generating superoxide anion and other ROS by NADPH oxidase-dependent mechanisms. This functional response, termed oxidative burst, contributes to host defense, but it can also result in collateral damage of host tissues. NADPH oxidase is a multicomponent enzyme system that catalyzes NADPH-dependent reduction of oxygen to superoxide anion. NADPH oxidase is activated by a variety of agents including N -formyl-methionyl-leucyl-phenylalanine (fMLP) and the protein kinase C (PKC) activator phorbol 12-myristate 13-acetate (PMA). These stimuli trigger biochemical cascades leading to the phosphorylation of several proteins of the NADPH oxidase system. In addition to the well-documented PKC pathway, one of these cascades involves activation of members of the mitogen-activated protein kinase (MAPK) family. Several studies have demonstrated that MAPK pathways such as extracellular signal- regulated kinases (ERK) 1/2 and p38 MAPK are activated in human neutrophils. Taurine is the most abundant free amino acid in most animal tissues and plays an important role in several essential biological processes (Huxtable 1992 ). A large number of reports have demonstrated the key role of taurine and its derivatives in the innate immune response (Schuller-Levis and Park 2004 ). It is widely recognized that taurine and related compounds such as hypotaurine and taurine chloramine exert a regulatory role in acute infl ammation. The protection by taurine and its derivatives on infl ammatory injury may be due to modulation of NADPH oxidase activity. It is noteworthy that taurine chloramines decrease PMA-stimulated superoxide production in human neutrophils by inhibiting phosphorylation of subunits of NADPH oxidase, eventually blocking the assembly of NADPH oxidase complex. Recently, it has been shown that thiotaurine (2-aminoethane thiosulfonate), a biomolecule structurally related to hypotaurine and taurine, prevents spontaneous apoptosis of human neutrophils (Capuozzo et al. 2013 ) and counteracts the damaging effect of oxidants in diabetic rat (Budhram et al. 2013 ). Interestingly, thiotaurine contains a sulfane sulfur that can be released as hydrogen sulfi de (H 2 S). It has been shown that H 2 S plays relevant roles, modulating several pathophysiological processes, including infl ammation. Taken together, these observations raise the possibility that thiotaurine, analogously to taurine and its derivatives, could modulate neutrophil activation. Thiotaurine is a thiosulfonates (RSO 2 SH) which has been occasionally detectedamong the products of biochemical reactions involving sulfur compounds. Thiotaurine is a metabolic product of cysteine in vivo and is produced by a spontaneous transsulfuration reaction involving thiocysteine (RSSH) and hypotaurine (RSO2H). Moreover, a sulfurtransferase which catalyzes the transfer of sulfur from mercaptopyruvate to hypotaurine with production of thiotaurine has been also reported. In the present study, thiotaurine has been assessed for an activity on functional response of human neutrophils. The results reveal that thiotaurine modulates fMLP- and PMA-mediated activation of human neutrophils, by inhibiting total ROS generation and superoxide anion production. Compared with fMLP-activated neutrophils, PMA-activated neutrophils were more susceptible to thiotaurine inhibition, suggesting that thiotaurine may interfere with the PKC-dependent pathway of neutrophil activation