The small, basic, and cysteine-rich antifungal protein PAF is abundantly secreted into the supernatant by the ␤-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 large number of small, basic, cysteine-rich antimicrobial proteins are produced by organisms throughout all kingdoms. They display a great variety in their primary structure, in species specificity, and in the mechanism of action.Few ascomycetes secrete strongly related antifungal proteins, which do not show any sequence homology with other antimicrobial proteins, but most of these proteins exhibit structural similarities (46): a net positive charge and numerous cysteine residues that are involved in disulfide bond formation. These properties contribute to a compact tertiary structure and a high stability against environmental impact and finally support the model of a membrane-disturbing nature. The antifungal protein PAF from P. chrysogenum and AFP from A. giganteus are the two most intensively studied peptides in the group of antifungals from ascomycetes, but the information available on their exact mechanism of action is still rather limited (32,55,68,69). PAF inhibits the growth of various important plant pathogenic and zoopathogenic filamentous fungi, e.g., Aspergillus fumigatus, A. niger, A. nidulans, and Botrytis cinerea. Previous studies revealed the induction of multifactorial detrimental effects on target organisms that include growth inhibition, reduction of cellular metabolism, severe changes in hyphal morphology, increased K ϩ efflux, and the generation of intracellular reactive oxygen species (ROS) (32, 48). PAF was found to...

The role of elevated intracellular calcium concentration ([Ca2+]) in activating calcium release from the sarcoplasmic reticulum (SR) was studied in skeletal muscle fibers microinjected with strong calcium buffers. After the injection of 3.8 +/- 0.5 mM (mean +/- S.E. of mean, n = 16) BAPTA (1,2-bis[o-aminophenoxy]ethane- N,N,N',N'-tetraacetic acid) or 2.2-2.8 mM fura-2 the normal increase in [Ca2+] during a depolarizing pulse was virtually eliminated. Even though calcium was released from the SR the kinetics of this release were markedly altered: the extensive buffering selectively eliminated the early peak component of SR calcium release with no effect on the maintained steady level. Microinjections of similar volumes but with low concentrations of fura-2 had no significant effect on the release waveform. The calcium released by voltage-dependent activation during depolarization may thus be involved in activating further calcium release, that is, in a calcium-induced calcium release mechanism.

Asymmetric membrane currents and fluxes of Ca 2+ release were determined in skeletal muscle fibers voltage clamped in a Vaseline-gap chamber. The conditioning pulse protocol 1 for suppressing Ca z+ release and the "hump" component of charge movement current (I~), described in the first paper of this series, was applied at different test pulse voltages. The amplitude of the current suppressed during the ON transient reached a maximum at slightly suprathreshold test voltages (-50 to -40 mV) and decayed at higher voltages. The component of charge movement current suppressed by 20 I~M tetracaine also went through a maximum at low pulse voltages. This anomalous voltage dependence is thus a property ofI,, defined by either the conditioning protocol or the tetracaine effect. A negative (inward-going) phase was often observed in the asymmetric current during the ON of depolarizing pulses. This inward phase was shown to be an intramembranous charge movement based on (a) its presence in the records of total membrane current, (b) its voltage dependence, with a maximum at slightly suprathreshold voltages, (c) its association with a "hump" in the asymmetric current, (d) its inhibition by interventions that reduce the "hump," (e) equality of ON and OFF areas in the records of asymmetric current presenting this inward phase, and (f) its kinetic relationship with the time derivative of Ca release flux. The nonmonotonic voltage dependence of the amplitude of the hump and the possibility of an inward phase of intramembranous charge movement are used as the main criteria in the quantitative testing of a specific model. According to this model, released Ca 2÷ binds to negatively charged sites on the myoplasmic face of the voltage sensor and increases the local transmembrane potential, thus driving additional charge movement (the hump). This model successfully predicts the anomalous voltage dependence and all the kinetic properties of I~ described in the previous papers. It also Address reprint requests to Dr.

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. Muscle contraction is activated by Ca 2ϩ release from the sarcoplasmic reticulum in response to plasma membrane depolarization. This process, called excitation-contraction coupling, takes place at the skeletal muscle triad junction, where T-tubules and the sarcoplasmic reticulum terminal cisternae are in close contact (1). Calcium release occurs via the calcium release complex, a macromolecular complex specifically localized in the skeletal muscle triad (2). The main components of this calcium release complex are the ryanodine receptor (RyR) 2 and the dihydropyridine receptor (DHPR) (3), both of which are calcium channels. It is now clear that a number of proteins are associated with these two calcium channels to form the calcium release complex, triadin being part of them. Triadin is an integral membrane protein of the sarcoplasmic reticulum, first identified in rabbit skeletal muscle as a 95-kDa glycoprotein specifically located in the triads (4, 5). Because of its co-localization with RyR in the triads, involvement of triadin in excitation-contraction coupling has been postulated (6, 7). Protein interaction studies have shown that the major molecular partners of triadin are RyR (8 -10), calsequestrin (CSQ), a protein that traps calcium inside the sarcoplasmic reticulum (11-13), and junctin (14). Functional studies have shown that triadin by itself r...

Background: A atoxins are major contaminants of feed used in poultry industry that negatively affect animal and human health. In Ethiopia, previous studies on a atoxins mainly considered cattle feed and milk, but scarce information exists for poultry feeds. Method: The aim of this study was to determine the burden of a atoxin in poultry feed in bishoftu.Cross sectional study was conducted from December, 2018 to May, 2019and 33 compound poultry feed samples were randomly collected from chicken rearing villages of Bishoftuand analyzed for G2, G1, B2 , B1 and total a atoxins using HPLC. Results: The result indicated thatfrom a total of 33 samples 31(94%) samples were contaminated with a atoxin. The mean level of a atoxin G2, G1, B2, B1 and total a atoxinswere 18.00 µg/g, 88.5499 µg/g, 13.50µg/g, 70.11µg/g and 190.18µg/g respectively. This study curtained the level of a atoxinin 25 (72.75%) samples for AFT and 22 (66.67%) samples for AFB1 were above the limit of FDA regulatory levels of 20µg/g for poultry feed. Conclusion: The study showed the high contamination of a atoxins in poultry feed. The study warrants the need for preventive strategies of a atoxin contamination including implementation of regulatory legislation in poultry feeds in Bishoftu.