Resuscitation of amebae deprived of essential symbiotes: Micrurgical studies*

SYNOPSIS. The effect of depletion and restoration of obligatory bacterial endosymbiotes on Amoeba proteus strain xD was studied. Removal of the symbiotes by culturing the amebae at 26.5 C resulted in loss of viability of the host cells, indicating that this strain is dependent on its endosymbiotes for survival. Amebae depleted of bacteria could initially be resuscitated by injection of isolated symbiotes, but prolonged deprivation led to irreversible changes. Nuclei of aposymbiotic amebae were viable when transplanted into the cytoplasm of normal cells, but the symbiote-depleted cytoplasm of heat-treated amebae could not be resuscitated by renucleation. No immediate ultrastructural changes were detected in aposymbiotic amebae except for clumping of nucleoli. Thus it appears that the symbiote performs an essential function as a cytoplasmic constituent.     

Subcellular localization of Cd in the root cells of<Emphasis Type="Italic">Allium sativum</Emphasis> by electron energy loss spectroscopy

The ultrastructural investigation of the root cells ofAllium sativum L. exposed to three different concentrations of Cd (100 (AM, 1 μM and 10 mM) for 9 days was carried out. The results showed that Cd induced several significant ultrastructural changes — high vacuolization in cytoplasm, deposition of electron-dense material in vacuoles and nucleoli and increment of disintegrated organelles. Data from electron energy loss spectroscopy (EELS) revealed that Cd was localized in the electron-dense precipitates in the root cells treated with 10 mM Cd. High amounts of Cd were mainly accumulated in the vacuoles and nucleoli of cortical cells in differentiating and mature root tissues. The mechanisms of detoxification and tolerance of Cd are briefly explained.     

Aflatoxin B1-induced ultrastructural alterations in matureZea mays embryos

Mature maize (Zea mays L.) embryos were exposed to aflatoxin B₁ (AFB₁) concentrations ranging from 0.1 to 25 µg/ml for 9 days. With increasing toxin concentration above 2 µg/ml, primary root elongation of germinated embryos was progressively inhibited, to reach a maximum value of 81% at 25 µ/ml toxin. An ultrastructural investigation of the subcellular alterations induced following toxin exposure provided evidence of deteriorative changes in several compartments of the plant cell. Alteration in membrane integrity (e.g., the tonoplast, plasmalemma and inner mitochondrial membrane) was a frequent feature of many cells. Apparent fusion of vacuoles, incorporation of cytoplasmic components into vacuoles and intravacuolar membrane whorls might be interpreted as deteriorative alterations. The results are discussed in the light of ultrastructural findings for other plant systems exposed to similar AFB₁ concentrations, as well as findings for animal systems.     

Cytotoxicity of Synthetic Fuel Products on Tetrahymena pyriformis. II. Shale Oil Retort Water*†‡

SYNOPSIS. Shale oil retort water is obtained by centrifuging the oil/water emulsion produced by oil shale retorting. The ciliate Tetrahymena pyriformis was exposed to retort water; 2, 1, and 0.5% initially increased motility; longer exposures decreased motility. Three, 4, and 5% all decreased motility. Cell lysis was directly related to concentration; after 24 h, population densities were 0, 10, and 25% of controls for 2, 1, and 0.5% retort water, respectively. Oxygen consumption paralleled the motility pattern: at lower concentrations it increased initially but decreased with extended exposures while at higher concentrations it decreased rapidly. The most striking cytologic alteration of cells exposed to the toxicant occurred in the membranes; alterations of mucocysts and glycogen content were also observed, but mitochondrial changes were not. Population growth was affected at much lower concentrations than the other test indices. The growth of test populations reached a plateau at values inversely related to concentration: concentrations <0.4% had no effect on growth rate.     

Oxidative stress by <Emphasis Type="Italic">Helicobacter pylori</Emphasis> causes apoptosis through mitochondrial pathway in gastric epithelial cells

Helicobacter pylori is a gram negative bacterium that infects the human stomach of approximately half of the world’s population. It produces oxidative stress, and mitochondria are one of the possible targets and the major intracellular source of free radicals. The present study was aimed at determining mitochondrial alterations in H. pylori-infected gastric epithelial cells and its relationship with oxidative stress, one of the recognized causes of apoptotic processes. Cells were treated with a strain of H. pylori for 24 h. Cellular oxidative burst, antioxidant defense analysis, mitochondrial alterations and apoptosis-related processes were measured. Our data provide evidence on how superoxide acts on mitochondria to initiate apoptotic pathways, with these changes occurring in the presence of mitochondrial depolarization and other morphological and functional changes. Treatment of infected cells with Vitamin E prevented increases in intracellular ROS and mitochondrial damage consistent with H. pylori inducing a mitochondrial ROS mediated programmed cell death pathway.     

Verticillium dahliae toxin induced alterations of cytoskeletons and nucleoli in Arabidopsis thaliana suspension cells

Summary. In plant cells, cytoskeletons play important roles in response to biotic and abiotic stresses. However, little is known about the dynamics of cytoskeletons when cells are attacked by unphysical stress factors such as elicitors and toxins. We report here that the toxin of Verticillium dahliae (VD toxin) induced changes of microfilaments (MFs) and microtubules (MTs) in Arabidopsis thaliana suspension-cultured cells. When cells were treated with a low concentration of VD toxin, MFs were disrupted ordinally from the cortex to the perinuclear region, and then recovered spontaneously; but the MTs persisted. The MFs in the perinuclear region showed more resistance to VD toxin than the cortical ones. In contrast, when cells were treated with a high concentration of VD toxin, MFs and MTs were disrupted sooner and more severely and did not recover spontaneously. Treatments with high concentrations of VD toxin also induced changes of nucleoli. At the early stages of treatment, a nucleus had a single ring-shaped nucleolus. At the later stages, multiple smaller and more brightly fluorescing nucleoli emerged in a single nucleus. Disrupted MFs could be recovered by removing the VD toxin before the ringshaped nucleoli appeared. All these results showed that MFs and MTs play important roles in the early defense responses against VD toxin in Arabidopsis suspension cells. The cytoskeletons may be used as sensors and effectors monitoring the defense reactions. The changes of nucleoli induced by VD toxin should be important characteristics of cell death. Correspondence and reprints: Department of Plant Sciences, College of Biological Sciences, China Agricultural University, Beijing 100094, People’s Republic of China.     

Ultrastructural alterations in response to zinc and nickel stress in the root cells of pigeonpea

Ultrastructure of root cortical cells of two genotypes of pigeonpea (Cajanus cajan), namely LRG30 and ICPL87, were grown under different concentrations of zinc and nickel. Growth reduction was found in roots. Electron micrographs of the root cortex cells of the two pigeonpea genotypes exposed to zinc and nickel showed alterations both at the cellular and the organelle level. At the lower concentrations, appearance of unusual deposition of electron dense globules in the vacuoles of the root cortical cells suggests that they may be involved in metal detoxification. Some of the cortical cells showed two nucleoli. The ultrastructural alterations are discussed in relation to the tolerance indices of the two genotypes.     

Electron microscope studies of experimentalEntamoeba histolytica infection in the guinea pig

Early cellular and vascular changes in response to invasion of lamina propria byEntamoeba histolytica were studied sequentially, at the ultrastructural level, in germfree guinea pigs inoculated intracecally with amebae and enteric flora derived from patients with acute amebic colitis. Approximately one week post-inoculation the animals developed acute colitis with mucosal invasion by trophic amebae. Although epithelial cells at the sites of amebic invasion showed progressive cytoplasmic changes and desquamation resulting in microerosions, most mesenchymal elements in the lamina propria appeared normal without cytopathic changes even when in direct contact with invading amebae. Only the polymorpho-nuclear leukocytes (PMN) apposed or topographically close to amebae exhibited degenerative changes which were characterized by condensation of nucleoplasm and cytoplasm, extracellular release of cytoplasmic components including granules, and, finally, lysis of cell membranes. Capillaries and venules in the lamina propria showed a variety of changes such as swelling and gap formation at the intercellular endothelial junctions and more rarely at the fenestrae. Blood vessels physically close to amebae showed formation of endothelial cytoplasmic blebs which pinched off into the vascular or extravascular space. Platelet and fibrin thromboses were common in the more severely damaged capillaries and venules. Fragments or clumps of fibrin-like material were found also in the extracellular spaces. Amebic invasion of the lamina propria, then, is accompanied by continued epithelial shedding, PMN degeneration, and changes in both capillaries and venules consisting of endothelial damage and occlusive thrombosis. The vascular changes appeared to be closely related to PMN degeneration resulting from interaction of PMN with invading amebae.     

Effect of fenitrothion on Spodoptera exigua larval development and ultrastructure of follicle cells

The effect of four sublethal concentrations (from 0.01165 to 0.3025 μg per insect, responding to LC₁₀/10, LC₁₀, LC₃₀ and LC₅₀) of the organophosphorus insecticide, fenitrothion, on Spodoptera exigua (Lepidoptera, Noctuidae) larval development and ultrastructure of follicular cells of adult female moths was tested. The most prominent malformations were: breaks in the cuticle, increased mortality during larval/pupal molting, alterations of nuclear envelope, changed heterochromatin pattern and abnormalities in mitochondrial ultrastructure. Some of the alterations are dose-dependent, but the other ones, like organization of rough endoplasmic reticulum (RER), show non-linear response: cells exposed to lower concentrations (LC₁₀/10 and LC₁₀) of the insecticide possessed clusters of accumulated RER, which were absent within the cells exposed to higher ones (LC₃₀ and LC₅₀). Formation of the eggshell was also markedly postponed within the groups exposed to lower doses. These findings prove that even minute amounts of xenobiotics may cause significant changes within exposed populations.     

Subcellular localization of cadmium in the root cells of<Emphasis Type="Italic">Allium cepa</Emphasis> by electron energy loss spectroscopy and cytochemistry

The ultrastructural investigation of the root cells ofAllium cepa L. exposed to 1 mM and 10 mM cadmium (Cd) for 48 and 72 h was carried out. The results indicated that Cd induced several obvious ultrastructural changes such as increased vacuolation, condensed cytoplasm with increased density of the matrix, reduction of mitochondrial cristae, severe plasmolysis and highly condensed nuclear chromatin. Electron dense granules appeared between the cell wall and plasmalemma. In vacuoles, electron dense granules encircled by the membrane were aggregated and formed into larger precipitates, which increase in number and volume as a consequence of excessive Cd exposure. Data from electron energy loss spectroscopy (EELS) confirmed that these granules contained Cd and showed that significantly higher level of Cd in vacuoles existed in the vacuolar precipitates of meristematic or cortical parenchyma cells of the differentiating and mature roots treated with 1 mM and 10 mM Cd. High levels of Cd were also observed in the crowded electron dense granules of nucleoli. However, no Cd was found in cell walls or in cells of the vascular cylinder. A positive Gomori-Swift reaction showed that small metallic silver grains were abundantly localized in the vesicles, which were distributed in the cytoplasm along the cell wall.     

Mitochondrial Dysfunction Enhances Susceptibility to Oxidative Stress by Down-Regulation of Thioredoxin in Human Neuroblastoma Cells

Increasing evidence suggests that Alzheimer’s disease is associated with mitochondrial dysfunction and oxidative damage. To develop a cellular model of Alzheimer’s disease, we investigated the effects of thioredoxin (Trx) expression in the response to mitochondrial dysfunction-enhanced oxidative stress in the SH-SY5Y human neuroblastoma cells. Treatment of SH-SY5Y cells with 15 mM of NaN₃, an inhibitor of cytochrome c oxidase (complex IV), led to alteration of mitochondrial membrane potential but no significant changes in cell viability. Therefore, cells were first treated with 15 mM NaN₃ to induce mitochondrial dysfunction, then, exposed to different concentrations of H₂O₂. Cell susceptibility was assessed by 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide assay and morphological observation. Expressions of Trx mRNA and protein were determined by RT-PCR; and Western-blot analysis, respectively. It was found that the SH-SY5Y cells with mitochondrial impairment had lower levels of Trx mRNA and protein, and were significantly more vulnerable than the normal cells after exposure to H₂O₂ while no significant changes of Trx mRNA and protein in SH-SY5Y cells exposed to H₂O₂ but without mitochondrial complex IV inhibition. These results, together with our previous study in primary cultured neurons, demonstrated that the increased susceptibility to oxidative stress is induced at least in part by the down-regulation of Trx in SH-SY5Y human neuroblastoma cells with mitochondrial impairment and also suggest the mitochondrial dysfunction-enhanced oxidative stress could be used as a cellular model to study the mechanisms of Alzheimer’s disease and agents for prevention and treatment.     

Effects of long-term starvation on hepatocyte ultrastructure of olive flounder Paralichthys olivaceus

The effects of nutritional conditions on alterations in condition factor, liver-somatic index, and hepatocyte ultrastructure in the olive flounder Paralichthys olivaceus were examined. Twelve weeks of starvation significantly decreased the condition factor and the liver-somatic index in the olive flounder. Hepatocytes underwent marked ultrastructural changes in response to 12 weeks of starvation. Compared to those of the initial control and fed group, the prominent features characterizing the hepatocytes of the starved group were: reduction in cell and nucleus size; apparent loss of nucleoli; condensation of chromatin; loss of stored glycogen; reduction of endoplasmic reticulum profile; increase in the number of electron-dense bodies containing large amounts of iron; and increased mitochondrial size. Results suggest that the histological changes caused by ultrastructural alterations in the hepatocytes can be used as alternative indicators to identify starvation in cultured P. olivaceus.     

Vital imaging of H9c2 myoblasts exposed to <Emphasis Type="Italic">tert</Emphasis>-butylhydroperoxide – characterization of morphological features of cell death

Background  

When exposed to oxidative conditions, cells suffer not only biochemical alterations, but also morphologic changes. Oxidative stress is a condition induced by some pro-oxidant compounds, such as by tert-butylhydroperoxide (tBHP) and can also be induced in vivo by ischemia/reperfusion conditions, which is very common in cardiac tissue. The cell line H9c2 has been used as an in vitro cellular model for both skeletal and cardiac muscle. Understanding how these cells respond to oxidative agents may furnish novel insights into how cardiac and skeletal tissues respond to oxidative stress conditions. The objective of this work was to characterize, through vital imaging, morphological alterations and the appearance of apoptotic hallmarks, with a special focus on mitochondrial changes, upon exposure of H9c2 cells to tBHP.     

Drug—induced alterations in the ultrastructural organization of Histoplasma capsulatum and Blastomyces dermatitidis

Aspects of the fine structure as seen in thin section of yeastlike cells ofHistoplasma capsulatum andBlastomyces dermatitidis exposed to polyenic antibiotics are described and illustrated by electron micrographs. The exposure of log phase yeastlike cells to minimal fungicidal concentrations of both amphotericin B (Fungizone) and hamycin resulted in detectable alterations of the plasma membrane, and, to a lesser extent, the mitochondria. WithH. capsulatum, ultrastructural changes were observed to occur within 1 h exposure to amphotericin B. Marked degenerative changes and plasmolysis were observed to occur within 6 hrs exposure of the yeastlike cells to both polyenes. The observed changes in ultrastructural appearance are compatible with the concept of binding of the polyene with membrane sterol and subsequent damage due to alterations of permeability.     

Dictyostelium discoideum surface changes elicited by high concentrations of cAMP

The effects of high concentrations of cAMP on both morphological and biochemical development of Dictyostelium discoideum amebae are reported. Observations using light and scanning electron microscopy (SEM) indicate that the cells' response to such treatment varies with the length of time they had been starved prior to cAMP addition. Vegetative and early developmental amebae become rounded within a short period after treatment. Such cells are capable of undertaking a normal aggregation after a delay of a few hours. A substantial induction of phosphodiesterase activity is elicited from these cells by cAMP treatment but their levels of cAMP surface binding sites remain low. cAMP addition to aggregation competent cells causes amebae first to flatten and then to retract into spherical forms and group into small aggregates. No induction of phosphodiesterase activity is observed in such cells and the levels of cAMP binding sites present on the amebae decrease rapidly. The data are discussed in terms of the different states of cAMP-sensitivity between vegative and aggregation-competent amebae.     

Cytotoxicity of synthetic fuel products on Tetrahymena pyriformis. II. Shale oil retort water.

Shale oil retort water is obtained by centrifuging the oil/water emulsion produced by oil shale retorting. The ciliate Tetrahymena pyriformis was exposed to retort water; 2, 1, and 0.5% initially increased motility; longer exposures decreased motility. Three, 4, and 5% all decreased motility. Cell lysis was directly related to concentration; after 24 h, population densities were 0, 10, and 25% of controls for 2, 1, and 0.5% retort water, respectively. Oxygen consumption paralleled the motility pattern: at lower concentrations it increased initially but decreased with extended exposures while at higher concentrations it decreased rapidly. The most striking cytologic alteration of cells exposed to the toxicant occurred in the membranes; alterations of mucocysts and glycogen content were also observed, but mitochondrial changes were not. Population growth was affected at much lower concentrations than the other test indices. The growth of test populations reached a plateau at values inversely related to concentration: concentrations less than 0.4% had no effect on growth rate.     

Cytolytic Activity of Naegleria fowleri Cell-free Extract1

ABSTRACT. The cytotoxic activity of a cell-free extract of Naegleria fowleri amebae on B103 rat nerve cells in culture was investigated. The cell-free extract was prepared by subjecting lysed amebae to centrifugation at 100,000 g for 1 h, precipitation of the supernatant fluid with 30–60% saturated ammonium sulfate, and desalting by group exclusion chromatography utilizing Sephadex G-25. The supernatant fluid recovered from this procedure was termed the soluble fraction. The Naegleria cytotoxic activity present in the soluble fraction was assayed by ⁵¹Cr released from labeled B103 cells. The Naegleria soluble fraction, when added to nerve cells, elicited blebs on the B103 target cell surface within 5 min after exposure to the fraction. Later, holes were observed in the B103 cell plasma membrane. These alterations were never observed on untreated B103 cells. Phospholipase A, phospholipase C, and protease activities were associated with the desalted ammonium sulfate-precipitable cytotoxic activity of N. fowleri cell-free lysate. The cytotoxic activity was impaired by ethylenediamine-tetraacetate (EDTA), phospholipase A inhibitor (Rosenthal's reagent), heating at 50°C for 15 min, or incubation at pH 10 for 60 min. Repeated freeze-thawing and inhibitors of proteolytic enzymes had no effect on the cytotoxic activity. Small amounts of ethanol (5% v/v) enhanced cytotoxic activity of the fraction. Phospholipases A and C, as well as other as yet unidentified cytolytic factors may be responsible for producing ⁵¹Cr release from target cells by the soluble fraction of N. fowleri extracts.     

Calcium amelioration of cadmium-induced cytotoxicity in cultured rat hepatocytes

Summary Parenchymal hepatocytes from neonatal rats were isolated, cultured about 24 h, exposed to cadmium with or without calcium, and processed for scanning electron microscopy. To assess the severity of cadmium-induced changes, exposed hepatocytes were categorized based upon the extent of morphological damage. Differences in surface blebbing, alterations in microvilli, variations in the degree of swelling, and changes in cell shape were used to categorize the severity of cell damage. A double-blind morphometric analysis (a geometricostatistical processing of two-dimensional data for the collection of three-dimensional information) of cellular changes was conducted for each exposure time and for each concentration of cadmium in the presence or absence of calcium. Significant decreases occurred in the percent relative volume of normal, flattened cells present in cultures exposed for 30 min to 50 or 100 μM cadmium in the absence of calcium. In contrast, the percent relative volume of severely damaged spherical cells was significantly increased after exposure to solutions containing 50 or 100 μM cadmium and lacking calcium. Percent relative volume of intermediate cells (which were slightly swollen and showed changes in microvillar number) was significantly increased following a 30 min exposure to all cadmium concentrations in the absence of calcium. The examination of hepatocytes exposed for 60 min showed that the degree of cadmium-induced cytotoxicity was more severe in the absence of calcium than was the case for the hepatocyte cultures exposed for 30 min: approximately 30% more spherical cells and 30% fewer flattened cells were present if cultures were exposed in the absence of calcium for 60 min compared to those exposed for 30 min. The degree of blebbing was significantly greater at all cadmium concentrations in the absence of calcium. The presence of calcium, therefore, reduced cadmium-induced cytotoxicity in primary cultures of rat hepatocytes subjected to morphometric analysis after scanning electron microscopy.     

Morphological study of the mammalian stress response: characterization of changes in cytoplasmic organelles, cytoskeleton, and nucleoli, and appearance of intranuclear actin filaments in rat fibroblasts after heat-shock treatment

Using both electron microscopy and immunological methods, we have characterized a number of changes occurring in rat fibroblasts after heat-shock treatment. Incubation of the cells for 3 h at 42 degrees-43 degrees C resulted in a number of changes within the cytoplasm including: a disruption and fragmentation of the Golgi complex; a modest swelling of the mitochondria and subtle alterations in the packing of the cristae; and alterations in cytoskeletal elements, specifically a collapse and aggregation of the vimentin-containing intermediate filaments around the nucleus. A number of striking changes were also found within the nuclei of the heat-treated cells: (a) We observed the appearance of rod-shaped bodies consisting of densely packed filaments. Using biochemical and immunological methods, these nuclear inclusion bodies were shown to be comprised of actin filaments. (b) Considerable alterations in the integrity of the nucleoli were observed after the heat-shock treatment. Specifically, there appeared to be a general relaxation in the condensation state of the nucleoli, changes in both the number and size of the granular ribonucleoprotein components, and finally a reorganization of the nucleolar fibrillar reticulum. These morphological changes in the integrity of the nucleoli are of significant interest since previous work as well as studies presented here show that two of the mammalian stress proteins, the major stress-induced 72-kD protein and the 110-kD protein, localize within the nucleoli of the cells after heat-shock treatment. We discuss these morphological changes with regards to the known biological and biochemical events that occur in cells after induction of the stress response.     

Ultrastructural Damage by Cadmium in a Marine Dinoflagellate,Prorocentrum micans1

Prorocentrum micans Ehrenberg, a free-living marine dinoflagellate, was used to test the intracellular toxic action of cadmium. The cells were cultivated in Erdschreiber medium, with Cd concentrations of 10–100 ppb. Thin sections of treated cells, examined ultramicroscopically, exhibited vacuolations, increased numbers of lysosomes, and severe mitochondrial damage. The first two alterations are a general response to toxicity; the third is Cd specific. Although some chloroplasts were affected by Cd, they were not very sensitive to its action. The nuclear apparatus was not morphologically affected.