László Csernoch

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.

Extracellular ATP and cancer: an overview with special reference to P2 purinergic receptors.

Purinergic signal transduction mechanisms have been appreciated as a complex intercellular signalling network that plays an important regulatory role in both short- and long-term processes in practically every living cell. One of the most intriguing aspects of the field is the participation of ATP and other purine nucleotides in the determination of cell fate and the way they direct cells towards proliferation, differentiation or apoptosis, thereby possibly taking part in promoting or preventing malignant transformation. In this review, following a very brief introduction to the historical aspects of purinergic signalling and a concise overview of the structure of and signal transduction pathways coupled to P2 purinergic receptors, the current theories concerning the possible ways how extracellular ATP can alter the function of tumour cells and the effectiveness of anticancer therapies are discussed, including pharmacological, nutritional, vasoactive and ‘anti-antioxidant’ actions of the nucleotide. The effects of ATP on animals inoculated with human tumours and on patients with cancer are looked over next, and then an overview of the literature regarding the expression and presumed functions of P2 purinoceptors on tumour cells in vitro is presented, sorted out according to the relevant special clinical fields. The article is closed by reviewing the latest developments in the diagnostic use of P2 purinergic receptors as tumour markers and prognostic factors, while discussing some of the difficulties and pitfalls of the therapeutic use of ATP analogues.

Intramembrane Charge Movement and L-Type Calcium Current in Skeletal Muscle Fibers Isolated from Control and mdx Mice

Dystrophin-deficient muscle fibers from mdx mice are believed to suffer from increased calcium entry and elevated submembranous calcium level, the actual source and functional consequences of which remain obscure. Here we compare the properties of the dihydropyridine receptor as voltage sensor and calcium channel in control and mdx muscle fibers, using the silicone-voltage clamp technique. In control fibers charge movement followed a two-state Boltzmann distribution with values for maximal charge, midpoint voltage, and steepness of 23 +/- 2 nC/ micro F, -37 +/- 3 mV, and 13 +/- 1 mV (n = 7). Essentially identical values were obtained in mdx fibers and the time course of charge recovery from inactivation was also similar in the two populations (tau approximately 6 s). In control fibers the voltage dependence of the slow calcium current elicited by 100-ms-long pulses gave values for maximal conductance, apparent reversal potential, half-activation potential, and steepness factor of 156 +/- 15 S/F, 65.5 +/- 2.9 mV, -0.76 +/- 1.2 mV, and 6.2 +/- 0.5 mV (n = 17). In mdx fibers, the half-activation potential of the calcium current was slightly more negative (-6.2 +/- 1.2 mV, n = 16). Also, when using longer pulses, the time constant of calcium current decay was found to be significantly larger (by a factor of 1.5-2) in mdx than in control fibers. These changes in calcium current properties are unlikely to be primarily responsible for a dramatic alteration of intracellular calcium homeostasis. They may be speculated to result, at least in part, from remodeling of the submembranous cytoskeleton network due to the absence of dystrophin.

Mitochondrial expression of the two-pore domain TASK-3 channels in malignantly transformed and non-malignant human cells

The presence of TASK-3 channels has been described in a number of healthy and malignantly transformed cells, showing mainly intracellular distribution with relatively insignificant labelling of the cell surface membrane. In this work, immunochemical and molecular biology methods were utilised to establish the intracellular organelle whose TASK-3 expression accounts for this strong intracellular labelling using cultured melanoma and HaCaT cells. Before the immunocytochemical experiments, the presence of TASK-3 mRNA was also confirmed in melanoma cells. Comparison of the results of the TASK-3- and mitochondrion-specific labelling indicated that the TASK-3 channel subunits were strongly expressed by mitochondria in both investigated cell types. Moreover, prominent TASK-3 expression of keratinocytes could also be demonstrated in histological sections excised from the human skin. These results indicate that TASK-3 channels are present in the mitochondria in both malignantly transformed and healthy cells, suggesting that they might have roles in ensuring mitochondrial functions.

Functional genomics of calcium channels in human melanoma cells

Ca2+‐signaling of human melanoma is in the focus of intensive research since the identification of the role of WNT‐signaling in melanomagenesis. Genomic and functional studies pointed to the important role of various Ca2+ channels in melanoma, but these data were contradictory. In the present study we clearly demonstrate, in a number of different ways including microarray analysis, DNA sequencing and immunocytochemistry, that various human melanoma cell lines and melanoma tissues overexpress ryanodine receptor type 2 (RyR2) and express P2X7 channel proteins as compared to melanocytes. These channels, although retain some of their usual characteristics and pharmacological properties, display unique features in melanoma cells, including a functional interaction between the two molecules. Unlike P2X7, RyR2 does not function as a calcium channel. On the other hand, the P2X7 receptor has an antiapoptotic function in melanoma cells, since ATP‐activation suppresses induced apoptosis, while knock down of the gene expression significantly enhances that.

Triadin Deletion Induces Impaired Skeletal Muscle Function

Triadin is a multiple proteins family, some isoforms being involved in muscle excitation-contraction coupling, and some having still unknown functions. To obtain clues on triadin functions, we engineered a triadin knock-out mouse line and characterized the physiological effect of triadin ablation on skeletal muscle function. These mice presented a reduced muscle strength, which seemed not to alter their survival and has been characterized in the present work. We first checked in these mice the expression level of the different proteins involved in calcium homeostasis and observed in fast muscles an increase in expression of dihydropyridine receptor, with a large reduction in calsequestrin expression. Electron microscopy analysis of KO muscles morphology demonstrated the presence of triads in abnormal orientation and a reduction in the sarcoplasmic reticulum terminal cisternae volume. Using calcium imaging on cultured myotubes, we observed a reduction in the total amount of calcium stored in the sarcoplasmic reticulum. Physiological studies have been performed to evaluate the influence of triadin deletion on skeletal muscle function. Muscle strength has been measured both on the whole animal model, using hang test or electrical stimulation combined with NMR analysis and strength measurement, or on isolated muscle using electrical stimulation. All the results obtained demonstrate an important reduction in muscle strength, indicating that triadin plays an essential role in skeletal muscle function and in skeletal muscle structure. These results indicate that triadin alteration leads to the development of a myopathy, which could be studied using this new animal model.

Intramembrane charge movement and sarcoplasmic calcium release in enzymatically isolated mammalian skeletal muscle fibres.

1 Single muscle fibres were dissociated enzymatically from the extensor digitorum longus and communis muscles of rats and guinea‐pigs. The fibres were mounted into a double Vaseline gap experimental chamber and the events in excitation–contraction coupling were studied under voltage clamp conditions. 2 The voltage dependence of intramembrane charge movement followed a two‐state Boltzmann distribution with maximal available charge of 26.1 ± 1.5 and 26.1 ± 1.3 nC μF−1, mid‐point voltage of –35.1 ± 5.0 and –42.2 ± 1.2 mV and steepness of 16.7 ± 2.2 and 17.0 ± 1.9 mV (means ±s.e.m., n= 7 and 4) in rats and guinea‐pigs, respectively. 3 Intracellular calcium concentration ([Ca2+]i) was monitored using the calcium‐sensitive dyes antipyrylazo III, fura‐2 and mag‐fura‐5. Resting [Ca2+]i was similar in rats and guinea‐pigs with 125 ± 18 and 115 ± 8 nm (n= 10 and 9), respectively, while the maximal increase for a 100 ms depolarization to 0 mV was larger in rats (6.3 ± 1.0 μm; n= 7), than in guinea‐pigs (2.8 ± 0.3; n= 4). 4 The rate of calcium release (Rrel) from the sarcoplasmic reticulum (SR) displayed an early peak followed by a fast and a slow decline to a quasi maintained steady level. After normalizing Rrel to the estimated SR calcium content (1.2 ± 0.1 and 0.9 ± 0.1 mm in rats and guinea‐pigs, respectively) and correcting for depletion of calcium in the SR the peak and steady levels at 0 mV, respectively, were found to be 2.50 ± 0.08 and 0.81 ± 0.06% ms−1 in rats and 2.43 ± 0.25 and 0.88 ± 0.01 % ms−1 in guinea‐pigs. The voltage dependence was essentially the same in both species, but different from that in amphibians. 5 These experiments show that enzymatic isolation yields functionally intact mammalian skeletal muscle fibres for Vaseline gap experiments. The data also suggest a close connection in the regulation of the different kinetic components of SR calcium release in mammalian skeletal muscle.

MEASUREMENTS OF INTRACELLULAR MG2+ CONCENTRATION IN MOUSE SKELETAL MUSCLE FIBERS WITH THE FLUORESCENT INDICATOR MAG-INDO-1

Measurements of intracellular free magnesium concentration ([Mg2+]i) were performed on enzymatically isolated skeletal muscle fibers from mice, using the fluorescent ratiometric indicator mag-indo-1. An original procedure was developed to calibrate the dye response within the fibers: fibers were first permeabilized with saponin in the presence of a given extracellular magnesium concentration and were then embedded in silicone grease. The dye was then pressure microinjected into the saponin-permeabilized silicone-embedded fibers, and fluorescence was measured. The results show that for all tested [Mg2+], the value of the measured fluorescence ratio was higher than that found in aqueous solutions. Furthermore, the apparent binding curve that could be fit to the in vivo ratio data was shifted toward higher [Mg2+] by a factor of approximately 2. Using the in vivo calibration parameters, the mean resting [Mg2+]i was found to be 1.53 +/- 0.16 mM (n = 7). In an attempt to gain insight into the myoplasmic magnesium buffering capacity, we measured, together with mag-indo-1 fluorescence, the current elicited by the application of carbamylcholine (CCh) to the endplate of isolated fibers, in the presence of a high extracellular magnesium concentration. The results show that, under these conditions, a change in [Mg2+]i displaying a time course and amplitude qualitatively consistent with the CCh-induced inward current can be measured.

Interacting effects of capsaicin and anandamide on intracellular calcium in sensory neurones.

Capsaicin (100 nM to 1 μM) and anandamide (200 nM to 10 μM) caused a transient increase in fluorescence of fura-2 loaded cultured small trigeminal neurones of rats measured with a ratiometric technique. The percentage of cells responding to capsaicin at 100 nM, 330 nM and 1 μM was 47.4%, 45.3%, and 70.4%, respectively. Averaged peak value of fluorescense ratio (R) at 340 and 380 nm excitation was slightly dose dependent. Peaks of anandamide-induced transients were R = 0.2 at 200 nM and 0.16 at 10 μM. Near 40% of capsaicin-sensitive cells responded also to anandamide. Anandamide (200 nM) inhibited the capsaicin-induced calcium influx. The results suggest that anandamide increases intracellular calcium and inhibits capsaicin-evoked calcium transients.

Protein kinase C isozymes regulate proliferation and high cell density‐mediated differentiation in HaCaT keratinocytes

Abstract:  Protein kinase C (PKC) isoforms play pivotal roles in the regulation of differentiation of normal human epidermal keratinocytes (NHEK). In this study, we investigated the participation of the PKC system in the proliferation and high cell density‐induced differentiation of the human immortalized keratinocyte line HaCaT. HaCaT keratinocytes possessed a characteristic PKC isoform pattern (PKCα, β, γ,δ,ε,η,θ,ζ), which altered during proliferation and differentiation. The GF109203X compound, a selective PKC inhibitor, suppressed the expressions of the late (granular cell) differentiation markers involucrin (INV) and filaggrin (FIL), and the terminal marker keratinocytes‐specific transglutaminase‐1 (TG), but did not affect the level of the early (spinous cell) marker keratin 10 (K10) and cellular proliferation. Phorbol 12‐myristate 13‐acetate (PMA), an activator of PKC, inhibited proliferation, elevated intracellular calcium concentration, decreased the expression of K10, and increased the expressions of INV, FIL, and TG. These data indicate that the endogenous activation of PKC regulates the expressions of the late differentiation markers, and that the exogenous activation of PKC by PMA results in the induction of terminal differentiation. Because the cellular effects of PMA were accompanied by differential down‐regulations of the sensitive PKC isoforms in proliferating and differentiating cultures, our findings argue for the differential roles of the existing PKC isoforms in the regulation of cellular proliferation and high cell density‐induced differentiation of HaCaT cells.