Abstract: When added to cells, a variety of autophagy inducers that operate through distinct mechanisms and target different organelles for autophagic destruction (mitochondria in mitophagy, endoplasmic reticulum in reticulophagy) rarely induce autophagic vacuolization in more than 50% or the cells. Here we show that this heterogeneity may be explained by cell cycle-specific effects. The BH3 mimetic ABT737, lithium, rapamycin, tunicamycin or nutrient depletion stereotypically induce autophagy preferentially in the G(1) and S phases of the cell cycle, as determined by simultaneous monitoring of cell cycle markers and the cytoplasmic aggregation of GFP-LC3 in autophagic vacuoles. These results point to a hitherto neglected crosstalk between autophagic vacuolization and cell cycle regulation.

Abstract: The present study investigated the accumulation of copper in various tissues and ultrastructural alterations in butterfish, Poronotus triacanthus. In acute toxicity test, butterfish were exposed to 250 μg/L copper for 24 h. In subacute toxicity test, fish were exposed to 25 μg/L copper for 7 days and then returned to normal water for 48 h. The levels of copper accumulation in the tissues were determined by using an atomic absorption spectrometer. After the 7 day exposure, the highest level of copper was found in the liver, followed by kidney, gills, and muscle tissues (3.64, 0.62, 0.59, and 0.34 μg/L, respectively). The recovery group has shown some reduction in copper level in these tissues when compared with those of the 7 day exposure group (3.09, 0.34, 0.30, and 0.27 μg/L, respectively). In gills, the major changes such as filament cell proliferation, increase in intercellular spaces, epithelial lifting, and thickening of the filament and lamellar epithelium were observed. In liver, the major changes such as swollen mitochondria, fragmented in rough endoplasmic reticulum, increases in number and size of lysosomes and lipid droplets. Infiltration of leukocytes, increasing hepatocyte size with pyknotic nuclei, and presence of vacuoles were also observed. In kidney, the changes included alterations of the first proximal tubules, as well as vacuolization of the cytoplasm, proliferation of lysosomes and mitochondria, dilation of endoplasmic reticulum, and finally, cell necrosis. The transmission electron microscopic analysis revealed that the 7 day exposure group had more severe effect in tissue alterations than the 24 h exposure group. Tissue regeneration was also observed in the 48 h recovery group. © 2007 Wiley Periodicals, Inc. Environ Toxicol 22: 92–100, 2007.

Abstract: This study presents morphological and biochemical evidence of programmed cell death (PCD) in Entamoeba histolytica induced by exposure of trophozoites to the aminoglycoside antibiotic G418. Morphological characteristics of PCD, including cell shrinkage, reduced cellular volume, nuclear condensation, DNA fragmentation and vacuolization were observed, with preservation of trophozoite membrane integrity. PCD is orchestrated biochemically by alterations in intracellular ion fluxes. In G418-treated trophozoites, overproduction of reactive oxygen species (ROS), decreased intracellular K+, increased cytosolic calcium, and decreased intracellular pH levels were observed. However, externalization of phosphatidylserine was not detected. These results suggest that amoebae can undergo PCD under stress conditions, and that this PCD shares several properties with PCD reported in mammals and in a variety of unicellular organisms.

Abstract: Cypermethrin was administered to Heteropneustes fossilis in chronic concentration to determine lesion of liver as indicators of tissue damage. The cypermethrin dose used was ¼ of 96 hr LC 50 . Histopathological changes in liver ranged from vacuolization, necrosis, fibrosis of perivascular region and disposition of yellow brown grains at different time of exposure viz; 20 th , 30 th , 40 th and 60 th days

Abstract: We examined how oxidative stress and cell damage develop in the liver of rats subjected to water-immersion stress (WIRS). In rats subjected to WIRS for 1.5, 3 or 6 h, serum alanine aminotransferase and aspartate aminotransferase activities increased time-dependently. In the liver tissue, vacuolization and apoptosis occurred at 1.5 h of WIRS and vacuolization further developed without further appearance of apoptosis at 3 h or 6 h. Serum lipid peroxide (LPO) and NOx (nitrite/nitrate) concentrations increased at 3 h of WIRS and these increases were enhanced at 6 h. In liver tissue, increases in LPO and NOx concentrations and myeloperoxidase activity and decreases in ascorbic acid and reduced glutathione concentrations and superoxide dismutase activity occurred at 3 h of WIRS and these changes were enhanced at 6 h, although vitamin E concentration and xanthine oxidase activity were unchanged. These results indicate that oxidative stress in the liver of rats with WIRS develops after the appearance of cell damage in the tissue, and suggests that oxidative stress is caused through disruption of the antioxidant defense system and increases in NO generation and neutrophil infiltration in the liver, which may contribute to the progression of cell damage in the tissue.

Abstract: Summary Background The ‘cosmeceutical’ agent 2-dimethylaminoethanol (DMAE) is a tertiary amine found in high concentration in numerous topical antiwrinkle preparations. Objectives We hypothesized that a 337 mmol L−1 (3%) DMAE reservoir applied to the skin could reproduce the cytopathology induced by other amines by maintaining a millimolar drug concentration within a certain depth of the skin layers, and that vacuolar cell expansion could account for the very rapid effect on the apparent skin fullness. Methods Morphological and functional assays were applied to cultured rabbit dermal fibroblasts treated with tertiary amines in vitro. A morphological verification of the vacuolization caused by topical DMAE was also attempted in vivo using the inner skin of the rabbit ear and in vitro using primary cultures of human cutaneous epithelial cells. Results Fibroblasts responded to DMAE (2·5–10 mmol L−1) by massive vacuolization (0·5–4 h; phase contrast observations). Triethanolamine, another chemical frequently used topically, was also active in this respect (10 mmol L−1). The vacuolar adenosine triphosphatase inhibitor bafilomycin A1 prevented DMAE- or triethanolamine-induced vacuolization; adding bafilomycin A1 or cell washout slowly reversed the established vacuolization induced by DMAE. Further effects of DMAE in cultured fibroblasts included a moderate cytotoxicity (10 mmol L−1) that was abated by bafilomycin A1 cotreatment, a concentration-dependent mitotic arrest (2·5 mmol L−1) and transient and mild effects on cell ploidy. The epidermis of the rabbit external ear was significantly thickened and exhibited clear perinuclear swelling indicative of vacuolization in response to 3% DMAE (1 h; paraffin tissue sections). Cultured human cutaneous epithelial cells responded to DMAE by vacuolization (inhibited by bafilomycin A1 cotreatment). Conclusions The vacuolar cytopathology induced by concentrated organic amines may be the cellular basis of the antiwrinkle effect of DMAE.

Abstract: Apoptotic changes in membrana granulosa of ovaries in goat (Capra hircus) have been studied in situ using light and scanning electron microscopy. Histologically, the degenerating granulosa cells were characterized by condensed cytoplasm, pyknotic nuclei and hazy cytosol. Most of the cytoplasmic components were condensed and were seen clumping against the pyknotic nucleus. The nucleus of degenerating cells stain darkly with giemsa while the cytoplasm were eosinophilic. In a few apoptotic cells heterochromatin bodies were seen adhering the nuclear membrane in the hyaline nucleosol. Under scanning electron microscopy apoptosis was marked by asymmetrical shrinkage and vacuolization of cytoplasm. The cell membrane of degenerating granulosa cells was smooth textured with a number of uneven depression and ruffles. Fragmentation of nucleus and pinching off of membrane bound nuclear fragments were the characteristic features observed in situ.

Abstract: Chlamydomonas reinhardtii and Anabaena cylindrica were used as model phytoplankton to show the ultrastructural changes in cell membranes and organelles exposed to the most common, commercial surfactants, the anionic sodium dodecyl sulphate and the cationic catamine, in a range of concentrations from 01 to 100 mg/l and an exposure range from 5 h to 7 days It was found that cell structure was not affected significantly by short-time exposure (to 5 h) to low concentrations of the surfactants, but they caused an intensification of cell vacuolization compared to the control The degree of change caused by the surfactants to the ultrastructure of cell organelles increased with the greater concentration of them At higher surfactant concentrations (30 to 50 mg/l), marked cell vacuolization was observed and there were changes to membrane structure, especially to the plasma membrane but also to the chloroplast thylakoids In addition, there were increases in the number and size of the mitochondria as well as morphological alterations to them There was also a reduction in the nuclear chromatin condensation At the highest surfactant concentrations (to 10 mg/ml), the cells had numerous degenerative changes, and death occurred to the majority of them Thus, the results of the model system revealed the precise dependence of the degree of structural reorganisation of the cell organelles on the concentration of surfactant in the culture medium The data on the state of the phytoplankton should be used for remotely monitoring the state of the microplanktonic organisms in water ecosystems

Abstract: Objective To study the death mode of human hepatocellular carcinoma Hep-3B cells and human lung adenocarcinoma A549 cells induced by bluetongue virus strain HbC3 （BTV-HbC3） and the mechanism of its action. Methods BTV-HbC3 was used to infect the tumor ceils, and the cytopathic effects （CPE） was observed. TUNEL staining was used to detect the apoptosis of tumor cells induced by BTV- HbC3. The changes of endoplasmic reticulum and nuclei treated with BTV-HbC3 were further examined by laser scanning confocal microscopy. The activities of caspase-3/7, caspase-8 and caspase-9 were determined by fluorescence analysis. Results Hep-3 B cells were sensitive to BTV-HbC3. Lots of early apoptotic cells were found by TUNEL staining. The laser scanning confocal microscopic examination showed characteristics of apoptosis, such as pyknotic nuclei, margination of nuclear chromatin and vacuolization of endoplasmic reticulumin in Hep-3B cells exposed to BTV-HbC3. The activity of caspase-3/7 was increased, but the activity changes of caspase-8 and caspase-9 were not found. A549 cells were sensitive to BTV-HbC3 too. But no apoptotic cells were observed by TUNEL staining. The results of laser scanning confocal microscopy showed marked vacuolization of endoplasmic reticulum, but chromatin margination was not found after ASd9 cells was exposed to BTV-HbC3. The activity of caspase-3/7 and caspase-9 was increased, but the activity of caspase-8 was not changed. Conclusion BTV-HbC3 induces apoptosis of Hep-3B tumor cells mainly through endoplasmic reticulum signal transduction pathway, and the features of cell death in A549 cells could be described as paraptosis. Key words: Bluetongue virus; Apoptosis; Paraptosis; Hep-3B cell; A549 cell

Abstract: It has been shown that deficit of serotonin during embryogenesis in rodents is accompanied by changes of morphological characteristics of neurons and glial cells at the period of postnatal development. A characteristic peculiarity of these changes is cell vacuolization that is of different expression in various cortical layers. In the experimental animals as compared with control ones, neurons of all neocortex layers have changed nuclei and a reduced volume of the cytoplasm. In neurons of upper layers, nuclei and cytoplasm contain occasional small vacuoles. In deep layers, vacuolization both of nuclei and of the cytoplasm is expressed to the much greater degree and vacuoles of large size are predominant. Results of immunocytochemical study have shown that in animals developing at the background of serotonin deficit there takes place a delay of the rates of formation and differentiation of astrocytic glia.

Abstract: The aim of the present study was to analyze the influence of a transgenically induced overexpression of the proinflammatory cytokine tumor necrosis factor-α (TNF) on potential degenerative or protective effects after infection with the neurotropic Borna Disease Virus (BDV). Additionally, the cytokine profile was measured in mice after BDV-infection with and without TNF-overexpression. Homo- (tg+/+) and heterozygous (tg+/-) transgenic animals with a moderate neuronal TNF-overexpression and non-transgenic neonatal animals with the same genetic background were infected intracerebrally with a mouse adapted BDV-strain. After necropsy, the mice brains were analyzed for the presence of pathohistological lesions. The viral nucleoprotein (BDV-N) expression was investigated immunhistologically. Furthermore, a possible influence on the promotor of the transgene via the BDV-infection was excluded by investigating the NR2B mRNA expression using real-time RT-PCR. Possible degenerative including apoptotic or necrotic cells or vacuolization were analyzed on hematoxylin and eosin-stained brain sections. In addition, vacuolization was examined by electron microscopy in detail. In addition, degenerative (Caspase 3, TUNEL-assay) and protective (p50, BDNF, ADNP) factors as well as TNF receptor 1 and 2 expression were investigated using immunohistochemistry. To study cellular activation, the nuclear diameter of GFAP-positive cells were determined 42 days after infection. Moreover, mRNA of IL-1α, IL-2, IL-4, IL-5, IL-6, IL-10, IL-12, TGFβ1, IFNγ, TNF, TNFR1, TNFR2 and BDNF was quantified by real time RT-PCR in the forebrain and selectively in Cortex cerebri, striatum, hippocampus and cerebellum. The viral protein BDV-N showed a disseminated distribution within the brain in all three BDV-infected mice groups starting at day 21 p.i. The inflammatory reaction correlated strongly with the transgenic status of the animals. Non-transgenic mice showed a mild cellular infiltration without progression during the investigation period. In contrast, the inflammatory reaction in both TNF-overexpressing transgenic mice groups caused a progressive, severe nonpurulent meningoencephalitis. In addition, activation of microglia and astrogliosis was observed. After BDV-infection, the total number of cells positive for p50, ADNP, TNFR1 and TNFR2 was increased in TNF-transgenic mice. Few apoptotic cells with features of activated astrocytes adjacent to inflammatory infiltrates in BDV-infected tg mice were detected. In transgenic BDV-infected mice, the amount of Caspase 3-positive and TUNEL-positive cells was significantly up-regulated. Surprisingly, most of the GFAP-positive cells showing apoptosis-like chromatin condensation lacked Caspase 3 immunoreactivity. However, neuronal necrosis, normally seen after BDV-infection, was not detected. Moreover, a moderate to severe vacuolization was noted in the brain beginning 35 days after BDV-infection. Interestingly, there was no significant difference between transgenic and non-transgenic BDV-infected mice. Electronmicroscopic examination revealed edema of the myelin sheath. Surprisingly, BDNF mRNA transcripts were significantly up-regulated in the hippocampus in hetero- and homozygous TNF-transgenic BDV-infected mice compared to non-infected and non-transgenic BDV-infected mice. However, there was no correlation with an increase of BDNF-positive cells after BDV-infection in the hippocampus. The basic levels of TNFto and TNFtg mRNA were approximately 2 fold higher in homozygous than in heterozygous animals and significantly increased compared to ntg mice. Interestingly, TNFtg induced an upregulation of host-derived TNF mRNA. After BDV-infection, no significant increase of TNFto mRNA values in transgenic mice was detected. However, TNFR1 and TNFR2 mRNA levels was significantly up-regulated until day 42 p.i. in all infected mice groups. This was more obvious in homozygous mice. After BDV-infection, the number of TNFR1- and TNFR2-positive cells increased significantly. This was most evident in both transgenic BDV-infected mice groups. This finding indicates that TNF-effects were mediated by increased receptor availability and not by further up-regulation of TNF itself. Elevated mRNA transcripts such as IL-1α, IL-6, IFNγ and IL-10 were measured in all BDV-infected mice compared to the non-infected control . However, total mRNA values were significantly higher in tg than in ntg mice. IL-2, IL-4, IL-5, IL-12 and TGFβ1 mRNA was not up-regulated after BDV-infection in any group. Though cytokine transcripts were more elevated in tg mice after BDV-infection, the general cytokine expression profile remained unchanged. The lack of IL-2 and IL-12 mRNA up-regulation indicates an incomplete Th1 response. This study shows that TNF-overexpression did not lead to a change in the cytokine profile after BDV-infection. However, a more pronounced and earlier upregulation of proinflammatory cytokine mRNA was detected. Furthermore, region specific neuroprotective effects in the hipoocampus as well as increased degenerative changes in the cortex cerebri and striatum were present. In conclusion, TNF-overexpression in combination with BDV-infection represents an excellent model to study further region-specific interactions of cytokines with neuroprotective factors and the antiviral immune response.

Abstract: The need for clinical awareness and diagnostic precision of glycogen storage disease type 2 (GSD2) has increased, as enzyme replacement therapy has become available. So far, only small series have reported the muscle pathology of late-onset GSD2. We reassessed 43 muscle biopsies of 38 GSD2 patients. In all patients the diagnosis of GSD2 has been established by biochemistry and/or mutational analysis of the GAA gene. Additionally to the expected morphological features, ultrastructural analysis revealed a high incidence of autophagic vacuoles, lipofuscin debris, structural Z-line disorganization and histological neurogenic-like pattern that were not thoroughly appreciated, previously. Comparing age at onset and morphology, excessive vacuolar and autophagic myopathy and mitochondrial disorganization of virtually all fibres is common in infants. At juvenile onset, a more moderate vacuolization without significant differences in overall morphology is notable. At late-onset, the spectrum of vacuolar myopathy is more divergent, ranging from almost normal to severe. Here pronounced secondary alterations are observed that include lipofuscin debris, autophagic vacuoles with residual lysosomal bodies and granular inclusions, structural mitochondrial and Z-line texture alterations. Moreover, there is a high incidence of subtle neurogenic-like alteration in all subtypes. Nineteen patients were genetically tested; in 15 patients the common leaky splicing mutation c.-45T>G (or IVS1-13T>G) in intron1 of the GAA gene was found on at least one allele, facilitating genetic screening. In our patients, GAA genotype appears not to be associated with secondary alterations such as autophagic vacuoles, structural alterations or neurogenic-like changes. These findings may have implications for our understanding of the pathogenesis of GSD2 and for assessing therapeutic success of enzyme replacement therapy.

Abstract: Autophagy is the unique, regulated mechanism for the degradation of organelles. This intracellular process acts as a prosurvival pathway during cell starvation or stress and is also involved in cellular response against specific bacterial infections. Vibrio cholerae is a noninvasive intestinal pathogen that has been studied extensively as the causative agent of the human disease cholera. V. cholerae illness is produced primarily through the expression of a potent toxin (cholera toxin) within the human intestine. Besides cholera toxin, this bacterium secretes a hemolytic exotoxin termed V. cholerae cytolysin (VCC) that causes extensive vacuolation in epithelial cells. In this work, we explored the relationship between the vacuolation caused by VCC and the autophagic pathway. Treatment of cells with VCC increased the punctate distribution of LC3, a feature indicative of autophagosome formation. Moreover, VCC-induced vacuoles colocalized with LC3 in several cell lines, including human intestinal Caco-2 cells, indicating the interaction of the large vacuoles with autophagic vesicles. Electron microscopy analysis confirmed that the vacuoles caused by VCC presented hallmarks of autophagosomes. Additionally, biochemical evidence demonstrated the degradative nature of the VCC-generated vacuoles. Interestingly, autophagy inhibition resulted in decreased survival of Caco-2 cells upon VCC intoxication. Also, VCC failed to induce vacuolization in Atg5−/− cells, and the survival response of these cells against the toxin was dramatically impaired. These results demonstrate that autophagy acts as a cellular defense pathway against secreted bacterial toxins.