COMPARISON OF MITOTIC PHENOMENA AND EFFECTS INDUCED BY HYPERTONIC SOLUTIONS IN HELA CELLS

Interphase HeLa cells exposed to solutions that are 1.6 x isotonic manifest a series of morphological transformations, several of which grossly resemble those which occur when untreated cells enter prophase. These include chromosome condensation with preferential localization at the nuclear envelope and nucleolus, ruffling of the nuclear envelope, and polyribosome breakdown. The nucleolus loses its fibrous component and appears diffusely granular. At 2.8 x isotonicity the nuclear envelope is selectively dispersed although other membranes show morphological alterations also. The characteristic transitions of the lysosomes, Golgi complex, and microtubules seen in normal mitosis do not occur during hypertonic treatment. All the changes induced with hypertonic solutions are rapidly reversible, and the nucleus particularly goes through a recovery phase which bears some similarity to that of the telophase nucleus. The prophase-like condensation of the chromatin following exposure of the intact cell to hypertonic medium cannot be reproduced on an ultrastructural level in the isolated nucleus with any known variation in salt concentration, suggesting significant modifications of the nuclear contents during isolation. In addition to these morphological responses, hypertonic solutions also markedly and reversibly depress macromolecular synthesis. The polyribosome disaggregation that results from exposure to hypertonic solutions may be partially prevented by prior exposure to elevated Mg⁺⁺ concentrations; this same ion is also partially effective in preventing the polyribosome breakdown which normally occurs as cells enter mitosis.     

Evidence for lysogeny and viral resistance in the cyanobacteriumPhormidium uncinatum

Escherichia coli B/5 12-h cultures were exposed to filter-sterilized acid mine water (AMW), fixed in situ, and examined for morphological changes by transmission electron microscopy, scanning electron microscopy, and x-ray spectrometry. Thin sections showed that layers of the Gram-negative envelope were altered and often lacking. Additionally, polar regions of the cell were frequently devoid of cytoplasm. AMW-exposed cells were distorted and had an amorphous substance associated with them. Spectra obtained by x-ray spectrometry suggested that this amorphous substance was cytoplasm rather than a mineral precipitate from AMW. Morphometric analyses of control and AMW-exposed populations showed significant differences in mean volume, length, and width of cells stressed in AMW; this indicates that smaller cells were selectively destroyed by the action of AMW. We concluded that loss of cytoplasm and cell lysis were the consequence of AMW damage to the bacterial envelope.     

Ultrastructural analysis of differentiation in Legionella pneumophila

Legionella pneumophila is an adaptive pathogen that replicates in the intracellular environment of fundamentally divergent hosts (freshwater protozoa and mammalian cells) and is capable of surviving long periods of starvation in water when between hosts. Physiological adaptation to these quite diverse environments seems to be accompanied by morphological changes (Gardu?o et al., p. 82-85, in Marre et al., ed., Legionella, 2001) and conceivably involves developmental differentiation. In following the fine-structural pathway of L. pneumophila through both in vitro and in vivo growth cycles, we have now discovered that this bacterium displays an unprecedented number of morphological forms, as revealed in ultrathin sections and freeze-fracture replicas for transmission electron microscopy. Many of the forms were identified by the obvious ultrastructural properties of their cell envelope, which included changes in the relative opaqueness of membrane leaflets, vesiculation, and/or profuse invagination of the inner membrane. These changes were best documented with image analysis software to obtain intensity tracings of the envelope in cross sections. Also prominent were changes in the distribution of intramembranous particles (clearly revealed in replicas of freeze-fractured specimens) and the formation of cytoplasmic inclusions. Our results confirm that L. pneumophila is a highly pleomorphic bacterium and clarify some early observations suggesting sporogenic differentiation in L. pneumophila. Since morphological changes occurred in a conserved sequence within the growth cycle, our results also provide strong evidence for the existence of a developmental cycle in L. pneumophila that is likely accompanied by profound physiological alterations and stage-specific patterns of gene expression.     

Expansion of the Candida albicans cell envelope in different morphological forms of the fungus.

Modes of cell envelope expansion were monitored in developing cells of Candida albicans 73/055 to which polystyrene beads were attached. Eight different conditions of culture medium, pH and temperature were used to promote growth in a variety of morphological forms. The cells were observed microscopically during growth in Sykes-Moore perfusion chambers, and sequential measurements of distances between the bead and the parent cell, and the bead and the apical tip were used to distinguish apical envelope expansion from general envelope expansion. Morphology index (Mi) was determined at each time point as an estimate of each cell's morphology. Calculations based on the measurements showed that general envelope expansion was inversely proportional to Mi, but that general expansion greater than 20% occurred only in cells with a final Mi less than 2.0, indicating that regulation of apical and general envelope expansion alone may be insufficient to determine the different morphologies seen in cells with higher Mi. The rate of expansion of the perimeter of cells was linearly proportional to the final Mi. This observation suggests that commitment to morphological development in C. albicans may in part involve commitment to a rate of envelope expansion, which itself helps determine the final morphology of a cell.     

Changes in the cellular structure of the salivary glands in Chironomus larvae resulting from mechanical cell damage

Rapid morphological changes were observed in some cells of hand-isolated salivary glands of Ch. thummi larvae. The nuclear envelope, routinely closely fitting the tightly packaged polytene chromosomes, was seen to lose its contact with the chromosomes and to attain a smooth round shape. Then unfolding of the chromosomes occurred, their banding patterns becoming clearly evident, probably through widening the interband regions; the chromosome length increased by about 20%. We argue that the changes observed were induced during gland isolation by lesions of the cell basal envelope in the sites of the fat body connections to the salivary gland.     

Cell Envelope Changes in Solvent-Tolerant and Solvent-Sensitive Pseudomonas putida Strains following Exposure to o-Xylene

Solvent-tolerant and -sensitive Pseudomonas putida strains were studied to determine their cell envelope changes following exposure to o-xylene. Both strains produced trans-unsaturated fatty acids. The tolerant strain showed an increase in total fatty acids, an increase in saturated fatty acids, and modified lipopolysaccharide. It is suggested that these envelope modifications aid in survival at high concentrations of organic solvents.     

VARIATIONS IN ENVELOPE MORPHOLOGY AND MINERALIZATION IN TRACHELOMONAS LEFEVREI (EUGLENOPHYCEAE)1

The mineralized envelope surrounding the trachelomonad cell is a complex of organic and inorganic components, anastomosed to form a structure that is characteristic of the species. Although there may be considerable morphological variability in envelope size, shape and ornamentation, its microarchitecture is characterized by two basic components: granular and needle-like deposits. The present study documents for the first time that manipulation of the culture conditions results in predictable changes in envelope ultrastructure, color and elemental composition. Supplemental iron (Fe) in the culture medium results in an increase of granular components in the envelope, whereas supplemental managanese (Mn) results in an increase of needle-like components; with nitrogen (N) supplements there is an increase of both granular and needle-like components in the envelope. It is also shown here that autoxidation of Fe may occur, resulting in the accumulation of Fe deposits along strands of unmineralized envelope mucilage. Autoxidation and deposition of Mn do not occur.     

Risk to preimplantation mouse embryos of combinations of heavy metals and radiation

The influence of arsenic, cadmium, lead or mercury on radiation risk to preimplantation mouse embryos in vitro was studied under various conditions. Morphological development, cell proliferation, and formation of micronuclei were used for assessment of risk after combined exposure to these metals and X-rays. No conditions were found under which arsenic altered radiation risk; the effects were merely additive. Cadmium acted similarly, though a few results indicated that morphological development might be impaired more strongly after combined exposure than expected from the addition of the single effects. Lead enhanced radiation risk with regard to micronucleus formation, but had an additive effect only in the case of morphological development and cell proliferation. Of all four metals, mercury had the greatest potential for enhancement of radiation risk, when morphological development and cell proliferation were studied. The observed combination effects exceeded even those effects which were calculated by taking into account the shape of the dose-effect curves (isobologram analysis, envelope of additivity). Mercury neither induced micronuclei nor enhanced their formation in combination experiments.     

Fine Structure of Vibrio cholerae During Toxin Production

The fine structural changes associated with cell growth and toxin production have been examined in Vibrio cholerae strain 569B. No morphological alterations in the cell envelope are apparent during logarithmic growth with thin-section techniques. However, internal swelling, suggesting alteration of cell envelope permeability, is evident particularly during the late logarithmic and early stationary phases of growth. Certain extracellular material demonstrable with negative-stain techniques does appear during the period of toxin production. The possible origin of this material is discussed. The effects of high temperature (37 C) and aeration on cell structure are also examined.     

Persistent expression of morphological abnormalities in the distant progeny of irradiated cells

The phenomenon of delayed heritable lethal damage (often referred to as ``lethal mutations') in the progeny of cells which survive irradiation is now well established, but little is known of the mechanism by which this cell death occurs. Current theories suggest a generalised genomic instability affecting all cells which leads to the production of some mutations which are lethal, or alternatively that a lethal mutation gene is activated, mutated or induced by radiation and leads to persistent and random cell death at high levels in the progeny. The aim of this study was to look at the morphology of progeny of irradiated cells at various times after irradiation to establish how widespread morphological abnormalities were in the population and whether there was any evidence that such abnormalities were clonal. Using two different cell lines, the results showed that morphological evidence possibly suggestive of apoptosis occurred in the cultures after all doses of radiation and up to 45 cell doublings after exposure. There was no evidence of a decrease in the numbers of damaged or dead cells in colonies with number of divisions after irradiation, or with decreasing original radiation dose. There was a significant dose-dependent increase in the number of cells with microvilli for both cell lines. The dose-dependency of this effect did not change with number of divisions after irradiation. It is clear that morphological evidence of cellular damage persists for several generations after the initial exposure. The effects are widespread in the cell population, and their constancy over time argues strongly for a general instability and against a clonal mechanism, since clonal descendants should die out and leave undamaged survivors. The lack of evidence for necrosis or senescence together with many morphological changes in the cultures suggestive of apoptosis could indicate an active mechanism of cell death. It is concluded that survivor populations of irradiated cells from two widely different mammalian cell lines demonstrate an altered phenotype including gross morphological changes. These result in a higher probability that cell division will fail to yield two healthy progeny. Received: 22 January 1996 / Accepted in revised form: 24 September 1996     

Survival of water stress in annual fish embryos: dehydration avoidance and egg envelope amyloid fibers

Diapausing embryos of Austrofundulus limnaeus survive desiccating conditions by reducing evaporative water loss. Over 40% of diapause II embryos survive 113 days of exposure to 75.5% relative humidity. An early loss of water from the perivitelline space occurs during days 1-2, but thereafter, rates of water loss are reduced to near zero. No dehydration of the embryonic tissue is indicated based on microscopic observations and the retention of bulk (freezable) water in embryos as judged by differential scanning calorimetry. Such high resistance to desiccation is unprecedented among aquatic vertebrates. Infrared spectroscopy indicates frequent intermolecular contacts via beta-sheet (14%) in hydrated egg envelopes (chorions). These beta-sheet contacts increase to 36% on dehydration of the egg envelope. Interestingly, the egg envelope is composed of protein fibrils with characteristics of amyloid fibrils usually associated with human disease. These features include a high proportion of intermolecular beta-sheet, positive staining and green birefringence with Congo red, and detection of long, unbranched fibrils with a diameter of 4-6 nm. The high resistance of diapause II embryos to water stress is not correlated with ontogenetic changes in the egg envelope.     

Comparative study of the effects of salinity and UV radiation on metabolism and morphology of the red macroalga <Emphasis Type="Italic">Acanthophora spicifera</Emphasis> (Rhodophyta,Ceramiales)

Increase of harmful radiation to the Earth’s surface due to ozone depletion results in higher exposure to harmful ultraviolet- B radiation (UV), while fluctuations in seawater salinity may alter water density, ionic concentration, nutrient uptake, and osmotic pressure. This study evaluated the effects of salinity and UV on metabolism and morphology of Acanthophora spicifera (M.Vahl) Børgesen. Water with 30 and 37 psu [g(salt) kg^–1(sea water)] was used for experiments during 7 d of exposure to UV (3 h per day). We demonstrated that UV treatment predisposed, irrespective of salinity, A. spicifera to a decrease in its growth rate and cell viability, as well as affected its morphological parameters. After exposure to PAR + UVA + UVB (PAB), samples showed structural changes and damage, such as increasing cell wall thickness and chloroplast disruption. Our results indicate that UV led to dramatic metabolic changes and cellular imbalances, but more remarkable changes were seen in samples exposed to high salinity.     

Effects of low-pH stress on the morphology and activity of bacteria from lakes receiving acid precipitation

Field and laboratory studies on the effects of low-pH stress are reported here for lacustrine bacteria. Two biochemical parameters, respiration and substrate utilization, reveal a marked decrease under severe low-pH stress. Bacterial ultrastructure and cell diversity can be correlated with a given level of pH. Structural changes in the cell envelope as a function of pH are highlighted. The biochemical, physiological and morphological observations are united into a hypothesis which seeks to relate cell adaptation to environmental stress.     

Chloride cell responses to long-term exposure to distilled and hard water in the gill of the armored catfish, Hypostomus tietensis (Loricariidae)

The morphological changes in the gill chloride cells of the armored catfish, Hypostomus tietensis , were investigated after 15 days' exposure to either distilled or hard water. The thickness of the water–blood barrier in the lamellae increased significantly in fish kept in distilled water due to the high proliferation of chloride cells. The apical surface of about 68% of chloride cells was sharply reduced by the development of an apical crypt with a sponge-like surface, although no change in the chloride cell fractional area was found. In contrast, H. tietensis kept in Na⁺, Cl⁻ and Ca²⁺ rich water displayed no significant changes in the number of chloride cells or in their apical surface morphology compared with the control fish. Chloride cell response to ion challenge in H. tietensis suggested the involvement of different strategies to maintain homeostasis in ion-poor water, which may be related to the life history of species.     

水稻淀粉胚乳细胞编程性死亡中细胞核变化特征

应用透射电子显微镜技术 ,观察了水稻 (OryzasativaL .)淀粉胚乳细胞编程性死亡过程中核的变化特征。伴随胚乳的发育进程 ,淀粉胚乳细胞核表现出衰退特征 :核变形、染色质凝缩、核膜多处被降解破坏、核基质外泄等。DNALadder显示核内大片段DNA呈严重的弥散状拖尾现象 ,而核内和胞质中在 14 0～ 180bp处有明显的条带。在核衰退的同时 ,其胞质中的粗面内质网、淀粉质体和线粒体等细胞器具有正常的代谢功能 ,细胞仍在合成并积累营养物质 ,淀粉胚乳细胞一边衰退一边行使其功能 ,直至死亡。这些结果表明 ,水稻淀粉胚乳在核衰退的同时 ,细胞仍在积极合成与积累贮藏产物 ,表现为一种特殊形式的植物细胞编程性死亡现象。此外 ,对淀粉胚乳细胞特有的核质关系、植物细胞编程性死亡过程中细胞核的变化等问题进行了讨论。     

Multidrug-resistance (MDR) phenotype of human osteosarcoma cells evaluated by quantitative morphological and electron microscopy analyses

Summary— Multidrug-resistant (MDR) variants of a human osteosarcoma cell line (U-2 OS) have been recently obtained by continuous exposure to doxorubicin (DX). The growth and phenotypic characteristics of these cell lines have been demonstrated to be related to the level of expression of P-glycoprotein. In this work, the morphological changes associated with MDR have been evaluated by quantitative image analysis and transmission electron microscopy. Resistant cells present morphological changes with respect to sensitive cells at both cytoplasmic and nuclear level. Some of these changes appear to be related to the degree of resistance but not to the direct presence of DX, since deprived cells maintain some modified characters, while others are partly lost. These findings suggest that DX exposure affects cell metabolism causing progressive changes of the cell morphotype.     

Hydrogen peroxide induces apoptotic-like cell death in coelomocytes of Themiste petricola (Sipuncula)

Apoptosis is an active form of cell death that plays a critical role in physiological and pathological conditions of multicellular organisms. These conditions include development, organogenesis, and elimination of infected, mutated, or damaged cells. Sipunculan cells may respond to changes in environmental exposure to oxidative stress by induction of apoptotic cell death. In coelomocytes of the sipunculan worm Themiste petricola, we evaluated morphological and biochemical changes that were induced by hydrogen peroxide (H2O2) and that could be compatible with an apoptotic-like phenotype. At an exposure of 100 mM H2O2, coelomocytes exhibited several morphological hallmarks of apoptosis such as chromatin condensation, nuclear segmentation, cell volume decrease, membrane blebbing, and formation of apoptotic bodies. Biochemical evidences of apoptotic-like cell death included exposure of phosphatidylserine (PS) in the outer leaflet of the plasma membrane and oligonucleosomal DNA fragmentation. In addition, exposure of coelomocytes to H2O2 induced a rapid massive loss of mitochondrial membrane potential and of the acidic pH of lysosomes. Overall, our results showed that, in sipunculan coelomocytes, H2O2 can induce changes compatible with an apoptotic-like phenotype. The finding of an oxidative-stress-induced apoptotic-like phenotype in a sipunculan worm may indicate that this kind of cell death process participates in regulation of cell number during physiological and pathological situations, including immune responses.     

An egg envelope component induces the acrosome reaction in sturgeon sperm

The acrosome reaction in Acipenser transmontanus sperm can be induced by a 66,000 dalton glycoprotein that is present in Layer 3 (L3) of the egg envelope and in egg water only following exposure of the eggs to fresh water. When egg water is fractionated on Sepharose CL 6B, the 66,000 dalton glycoprotein-containing fractions possess acrosome reaction inducing activity. Egg water may be species-specific in its ability to elicit the acrosome reaction, as demonstrated by the fact that it has no effect on the sperm of Acipenser fulvescens. Egg jelly possesses no acrosome reaction, inducing activity. The major carbohydrate-containing component of the egg envelope is L3, a layer that contains galactose residues. L3 possesses a 70,000 dalton glycoprotein prior to freshwater exposure and lacks the 66,000 dalton component. If isolated from polyacrylamide gels, the 70,000 dalton glycoprotein elicits acrosome reactions in what appears to be a species specific manner. After freshwater exposure, L3 contains both the 70,000 dalton glycoprotein and the 66,000 dalton glycoprotein that is also present in egg water. The appearance of the 66,000 dalton inducer can be blocked by the incubation of eggs in fresh water containing inhibitors of trypsin activity. Thus, the soluble inducer in egg water may be proteolytically derived from a higher molecular weight complex in the egg envelope.