Amatoxin poisoning is caused by mushroom species belonging to the genera Amanita, Galerina and Lepiota with the majority of lethal mushroom exposures attributable to Amanita phalloides. High mortality rate in intoxications with these mushrooms is principally a result of the acute liver failure following significant hepatocyte damage due to hepatocellular uptake of amatoxins. A wide variety of amatoxins have been isolated; however, α-amanitin (α-AMA) appears to be the primary toxin. Studies in vitro and in vivo suggest that α-AMA does not only cause hepatocyte necrosis, but also may lead to apoptotic cell death. The objective of this study was to evaluate the complex hepatocyte apoptosis in α-AMA cytotoxicity. All experiments were performed on primary cultured canine hepatocytes. The cells were incubated for 12 h with α-AMA at a final concentration of 1, 5, 10 and 20 μM. Viability test (MTT assay), apoptosis evaluation (TUNEL reaction, detection of DNA laddering and electron microscopy) were performed at 6 and 12 h of exposure to α-AMA. There was a clear correlation between hepatocyte viability, concentration of α-AMA and time of exposure to this toxin. The decline in cultured dog hepatocyte viability during the exposure to α-AMA is most likely preceded by enhanced cellular apoptosis. Our results demonstrate that apoptosis might contribute to pathogenesis of the severe liver injury in the course of amanitin intoxication, particularly during the early phase of poisoning.

The toadstool death cap (Amanita phalloides) and its subspecies, destroying angel (A. virosa) and death angel (A. verna) are responsible for nearly 95% of all fatal mushroom poisonings. High mortality rate in A. phalloides intoxications is principally a result of the acute liver failure following significant hepatocyte damage due to hepatocellular uptake of amanitins, the major toxins of this mushroom. This study evaluated early morphological and functional alterations in hepatocytes exposed to different concentrations of alpha-amanitin (alpha-AMA). All experiments were performed on cultured canine hepatocytes since intoxicated with A. phalloides dogs have clinical course and pathological findings similar to those seen in humans. The overall functional integrity and viability of cultured hepatocytes were assessed using the MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] assay and by measurements of lactate dehydrogenase (LDH), total protein, and urea levels. Our results showed that the course of alpha-AMA toxicity in cultured dog hepatocytes is divided into two phases. The first phase comprises functional cell impairments expressed by significant increase of LDH activity and inhibition of protein and urea synthesis when compared with the control group. This is followed by discrete changes in hepatocyte ultrastructure, including marginalization and condensation of nuclear chromatin, as well as formation of the foamlike cytoplasm. The second stage is lethal and is characterized by ongoing necrosis, and/or apoptosis. This may be related to dose of toxin and time of exposure.

This study aims at presenting histology of growing and mature antlers in red deer stag (Cervus elaphus). Growing antlers constitute a model organ for examining regeneration processes of tissues because they are the only mammalian appendages capable of regeneration. Histological study revealed that the tip of a growing antler consists of hairy skin, perichondrium, mesenchyme and chondroprogenitors area. By performing immunochistochemistry, we found that cell expressing Ki-67 and PCNA antigens were localized in basal layer of epidermis, skin glands and beneath their secretory sections, mesenchyme as well as within and in the vicinity of central blood vessels. Ultrastructurally, cells from chondroprogenitors zone have chondroblast-like morphology and take part in producing of collagen fibres followed by the process of cartilage mineralization. However, mature antlers also consist of lamellar osseous tissue.

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GABA type A receptors (GABA A Rs) belong to the pentameric ligand-gated ion channel (pLGIC) family and play a crucial role in mediating inhibition in the adult mammalian brain. Recently, a major progress in determining the static structure of GABA A Rs was achieved, although precise molecular scenarios underlying conformational transitions remain unclear. The ligand binding sites (LBSs) are located at the extracellular domain (ECD), very distant from the receptor gate at the channel pore. GABA A R gating is complex, comprising three major categories of transitions: openings/closings, preactivation, and desensitization. Interestingly, mutations at, e.g., the ligand binding site affect not only binding but often also more than one gating category, suggesting that structural determinants for distinct conformational transitions are shared. Gielen and co-workers (2015) proposed that the GABA A R desensitization gate is located at the second and third transmembrane segment. However, studies of our and others’ groups indicated that other parts of the GABA A R macromolecule might be involved in this process. In the present study, we asked how selected point mutations (β 2 G254V, α 1 G258V, α 1 L300V, and β 2 L296V) at the M2 and M3 transmembrane segments affect gating transitions of the α 1 β 2 γ 2 GABA A R. Using high resolution macroscopic and single-channel recordings and analysis, we report that these substitutions, besides affecting desensitization, also profoundly altered openings/closings, having some minor effect on preactivation and agonist binding. Thus, the M2 and M3 segments primarily control late gating transitions of the receptor (desensitization, opening/closing), providing a further support for the concept of diffuse gating mechanisms for conformational transitions of GABA A R.