Henrietta Szappanos

Antifungal Protein PAF Severely Affects the Integrity of the Plasma Membrane of Aspergillus nidulans and Induces an Apoptosis-Like Phenotype

ABSTRACT The small, basic, and cysteine-rich antifungal protein PAF is abundantly secreted into the supernatant by the β-lactam producer Penicillium chrysogenum. PAF inhibits the growth of various important plant and zoopathogenic filamentous fungi. Previous studies revealed the active internalization of the antifungal protein and the induction of multifactorial detrimental effects, which finally resulted in morphological changes and growth inhibition in target fungi. In the present study, we offer detailed insights into the mechanism of action of PAF and give evidence for the induction of a programmed cell death-like phenotype. We proved the hyperpolarization of the plasma membrane in PAF-treated Aspergillus nidulans hyphae by using the aminonaphtylethenylpyridinium dye di-8-ANEPPS. The exposure of phosphatidylserine on the surface of A. nidulans protoplasts by Annexin V staining and the detection of DNA strand breaks by TUNEL (terminal deoxynucleotidyltransferase-mediated dUTP-biotin nick end labeling) gave evidence for a PAF-induced apoptotic-like mechanism in A. nidulans. The localization of reactive oxygen species (ROS) by dichlorodihydrofluorescein diacetate and the abnormal cellular ultrastructure analyzed by transmission electron microscopy suggested that ROS-elicited membrane damage and the disintegration of mitochondria played a major role in the cytotoxicity of PAF. Finally, the reduced PAF sensitivity of A. nidulans strain FGSC1053, which carries a dominant-interfering mutation in fadA, supported our assumption that G-protein signaling was involved in PAF-mediated toxicity.

A purinergic signal transduction pathway in mammalian skeletal muscle cells in culture

Abstract. The effects of adenosine 5′-triphosphate (ATP) on human and mouse skeletal muscle fibres in primary culture were investigated. ATP-evoked changes in intracellular calcium concentration ([Ca2+]i) were measured and compared with those induced by agonists of the nicotinic acetylcholine (Ach)- and P2X purinoreceptors. While ATP was effective on both myoblasts and multi-nucleated myotubes in the micromolar range, Ach failed to induce any change in [Ca2+]i at early stages of development. In contrast, myofibres with peripheral nuclei showed little response to ATP but responded to Ach with a large change in [Ca2+]i. The responsiveness of the myotubes to Ach paralleled that to potassium. The removal of external calcium abolished the response to ATP. P2X receptor agonists mimicked the response to ATP with the order of potency being ATP>2′,3′-O-(4-benzoyl)-benzoyl-ATP>β,γ-methylene-ATP>α,β-methylene-ATP. Under voltage-clamp conditions ATP induced an inward current that showed little inactivation. These results are consistent with the existence of P2X receptor-mediated signal transduction pathway in cultured mammalian skeletal muscle cells.

The antifungal protein AFP secreted by Aspergillus giganteus does not cause detrimental effects on certain mammalian cells.

The antifungal protein AFP is a small, cystein-rich protein secreted by the imperfect ascomycete Aspergillus giganteus. The protein efficiently inhibits the growth of filamentous fungi, including a variety of serious human and plant pathogens mainly of the genera Aspergillus and Fusarium, whereas AFP does not affect the growth of yeast and bacteria. This restricted susceptibility range makes it very attractive for medical or biotechnological use to combat fungal infection and contamination. We, therefore, analyzed whether AFP affects the growth or function of a number of mammalian cells. Here we show that the protein neither provokes any cytotoxic effects on human endothelial cells isolated from the umbilical vein nor activates the immune system. Moreover, potassium currents of neurons and astrocytes do not change in the presence of AFP and neither excitatory processes nor the intracellular calcium homeostasis of cultured skeletal muscle myotubes are affected by AFP. Our data, therefore, suggest that AFP is indeed a promising candidate for the therapeutic or biotechnological use as a potential antifungal agent.

Altered elementary calcium release events and enhanced calcium release by thymol in rat skeletal muscle.

The effects of thymol on steps of excitation-contraction coupling were studied on fast-twitch muscles of rodents. Thymol was found to increase the depolarization-induced release of calcium from the sarcoplasmic reticulum, which could not be attributed to a decreased calcium-dependent inactivation of calcium release channels/ryanodine receptors or altered intramembrane charge movement, but rather to a more efficient coupling of depolarization to channel opening. Thymol increased ryanodine binding to heavy sarcoplasmic reticulum vesicles, with a half-activating concentration of 144 micro M and a Hill coefficient of 1.89, and the open probability of the isolated and reconstituted ryanodine receptors, from 0.09 +/- 0.03 to 0.22 +/- 0.04 at 30 micro M. At higher concentrations the drug induced long-lasting open events on a full conducting state. Elementary calcium release events imaged using laser scanning confocal microscopy in the line-scan mode were reduced in size, 0.92 +/- 0.01 vs. 0.70 +/- 0.01, but increased in duration, 56 +/- 1 vs. 79 +/- 1 ms, by 30 micro M thymol, with an increase in the relative proportion of lone embers. Higher concentrations favored long events, resembling embers in control, with duration often exceeding 500 ms. These findings provide direct experimental evidence that the opening of a single release channel will generate an ember, rather than a spark, in mammalian skeletal muscle.

Differential expression of purinergic receptor subtypes in the outer hair cells of the guinea pig

ATP acts as a neuro-modulator through purinoceptors in many different tissues. Many subtypes of these receptors have been identified in the inner ear, but so far only two types have been shown to be present in the membrane of the isolated outer hair cells (OHCs). The aim of this study was to detect and visualize the existence and distribution of purinoceptor subtypes as well as to study the [Ca(2+)](i) response of these cells in response to stimulation with ATP. Four P2X and three P2Y receptor subtypes were identified with different expression pattern in the membrane of guinea pig outer hair cells. Whereas intense labeling was observed for P2X1, P2X2, P2X4, P2Y1, P2Y2, and P2Y4, the labeling for the subtype P2X7 was weak. There was a marked difference in the distribution of the receptors along the surface of the cells with a homogenous distribution in cases of P2X1, P2X4, and P2Y1. In contrast, P2X2 and P2Y2 receptor density was high mainly at the apical, while P2X7 and P2Y4 at the basal pole of the cells. Similarly a heterogeneity was observed in the ATP-induced transient elevation in [Ca(2+)](i), which had either fast kinetics without desensitization or slow rise with desensitization.

Contribution from P2X and P2Y purinoreceptors to ATP-evoked changes in intracellular calcium concentration on cultured myotubes.

Although the alteration of purinoreceptor pattern on skeletal muscle is known to accompany physiological muscle differentiation and the pathogenesis of muscle dystrophy, the exact identity of and the relative contribution from the individual receptor subtypes to the purinergic signal have been controversial. To identify these subtypes in cultured myotubes of 5–10 nuclei, changes in intracellular calcium concentration and surface membrane ionic currents were detected and calcium fluxes calculated after the application of the subtype-specific agonists 2′3′-O-(benzoyl-4-benzoyl)-ATP (BzATP), 2-methyltio-ADP and UTP. The effectiveness of these agonists together with positive immunocytochemical staining revealed the presence of P2X4, P2X5, P2X7, P2Y1 and P2Y4 receptors. siRNA-reduced protein expression of P2X5, P2X7 and P2Y1 receptors was accompanied by reduction in the ATP-evoked calcium transients. Furthermore, anti-P2X7 siRNA caused a significant drop in the early peak and delayed steady component of the calculated calcium flux. The use of its antagonist, oxidized ATP, similarly to transfection with anti-P2X7 siRNA caused significant reduction in the agonist-elicited ionic currents IATP and IBzATP, with a greater drop in the latter. Our results demonstrate that the activation of ionotropic P2X4, P2X5 and P2X7 and metabotropic P2Y1 and P2Y4 purinoreceptors participates in forming the calcium transients of multinucleated myotubes.

Differential effects of maurocalcine on Ca2+ release events and depolarization-induced Ca2+ release in rat skeletal muscle.

Maurocalcine (MCa), a 33 amino acid toxin obtained from scorpion venom, has been shown to interact with the isolated skeletal‐type ryanodine receptor (RyR1) and to strongly modify its calcium channel gating. In this study, we explored the effects of MCa on RyR1 in situ to establish whether the functional interaction of RyR1 with the voltage‐sensing dihydropyridine receptor (DHPR) would modify the ability of MCa to interact with RyR1. In developing skeletal muscle cells the addition of MCa into the external medium induced a calcium transient resulting from RyR1 activation and strongly inhibited the effect of the RyR1 agonist chloro‐m‐cresol. In contrast, MCa failed to affect the depolarization‐induced Ca2+ release. In intact adult fibres MCa did not induce any change in the cytosolic Ca2+ concentration. However, when the surface membrane was permeabilized and calcium release events were readily observable, MCa had a time‐dependent dual effect: it first increased event frequency, from 0.060 ± 0.002 to 0.150 ± 0.007 sarcomere−1 s−1, and reduced the amplitude of individual events without modifying their spatial distribution. Later on it induced the appearance of long‐lasting events resembling the embers observed in control conditions but having a substantially longer duration. We propose that the functional coupling of DHPRs and RyR1s within a Ca2+ release unit prevents MCa from either reaching its binding site or from being able to modify the gating not only of the RyR1s physically coupled to DHPRs but all RyR1s within the Ca2+ release unit.

Differences in purinergic and voltage-dependent signalling during protein kinase Cα overexpression- and culturing-induced differentiation of C2C12 myoblasts

Differentiation of skeletal muscle cells both in vivo and in vitro is accompanied by the development of voltage-dependent processes and alterations in purinergic signalling. To date at least two independent methods have been used to induce differentiation in primary cultures, namely, appropriate modification of culturing conditions and overexpression of specific protein kinase C (PKC) isoenzymes. Here we characterize and compare the development of purinergic and depolarization-dependent alterations using these two methods to induce differentiation in C2C12 cells. We demonstrate that depolarization- and ATP-evoked Ca2+ responses underwent functional development during differentiation, and the characteristics of this progress were dependent on the actual differentiation-promoting stimulus. Overexpression of PKCα anticipated the appearance of robust increases in the intracellular calcium concentration upon ATP administration but failed to do so after depolarizing stimuli. Moreover, the first phase of the biphasic ATP-induced response observed in differentiated myotubes induced by culturing was not present in differentiated PKCα-overexpressing cells, suggesting that although purinergic signalling developed very early, purinergic stimuli failed to activate the voltage-dependent mechanisms of these cells even at subsequent stages of differentiation. Disruption of the coupling of purinergic signalling to depolarization-activated mechanisms may be explained by our observations that PKCα-overexpression changed the purinergic receptor pattern of immature myoblasts differently from what was seen in the course of culturing-induced differentiation. PKCα-specific alterations were characterized by the lack of increase in the expression of P2X7 receptors and the failure of P2Y4 receptors to appear and P2Y2 receptors to disappear. The effects of PKCα-overexpression were proven to be specific since the overexpression of the hyperproliferative isoenzyme PKCδ failed to induce any of the changes promoted by PKCα. Our data suggest that the method of inducing differentiation in skeletal muscle cells modifies not only the course of development but also the interaction of depolarization-dependent and purinergic pathways.