Survey of virally mediated permeability changes.

1. Sendai virus causes permeability changes when added to freshly isolated brain cells (cerebellum or ependymal cells) or to a culture of forebrain cells. 2. Sendai virus causes permeability changes when added to organ cultures of ferret lung or nasal turbinate. Influenza virus causes no permeability changes under these conditions. 3. Rabies virus and vesicular-stomatitis virus, in contrast with Sendai virus, do not cause permeability changes in BHK cells or Lettrée cells. 4. Serum from patients suffering from viral hepatitis does not cause permeability changes in human leucocytes; addition to Sendai virus causes permeability changes. 5. It is concluded that permeability changes accompanying viral entry occur only with certain types of paramyxovirus, but that there is little restriction on cell type. 6. MDBK cells infected with Sendai virus show permeability changes during viral release, similar to those that occur during viral entry. Because these changes do not appear to be restricted to paramyxoviruses, they may have considerable clinical significance.     

Crohn’s disease of the colon: ultrastructural changes in submuscular interstitial cells of Cajal

Interstitial cells of Cajal (ICC) at the submuscular border of the human colon (ICC-SMP) are the proposed pacemaker cells of the musculature. In patients with Crohn’s disease (CD) of the colon, ICC-SMP showed characteristic cytological changes from controls. The changes comprised secondary lysosomes in connection with lipid droplets and cytoplasmic vacuoles or multiple empty, confluent and often outbulging vacuoles merging with cisterns of granular endoplasmic reticulum and clusters of glycogen granules. These changes were most pronounced in patients with macroscopical mucosal inflammation but were also demonstrable in uninvolved colonic segments. Relationships of ICC to other cells were undisturbed. The changes were selective to ICC-SMP, as glial cells, muscle cells and fibroblast-like cells at the submuscular border showed no cytological alterations compared with controls. Varicosities of the submuscular plexus were often empty and dilated. Fibroblast-like cells selectively encased macrophages and mast cells. The cytological changes in ICC-SMP in CD are thus similar to changes seen in ulcerative colitis and may be of pathophysiological significance with regard to the motility and sensory disturbances seen in patients with CD.     

AFM-detected apoptotic changes in morphology and biophysical property caused by paclitaxel in Ishikawa and HeLa cells

The apoptosis of cancer cells is associated with changes in the important cell properties including morphology, surface roughness and stiffness. Therefore, the changes in morphology and biophysical properties can be a good way of evaluating the anticancer activity of a drug. This study examined the effect of paclitaxel on the properties of Ishikawa and HeLa cells using atomic force microscopy (AFM), and the relationship between the changes in morphology and the biophysical properties and apoptosis was discussed. The viability and proliferation of the cells were analyzed using the methylthiazol tetrazolium (MTT) method and a TUNEL assay to confirm cellular apoptosis due to a paclitaxel treatment. AFM observations clearly showed the apoptotic morphological and biophysical changes in Ishikawa and HeLa cells. After the paclitaxel treatment, the cell membrane was torn and holed, the surface roughness was increased, and the stiffness was decreased. These changes were observed more apparently after a 24 h treatment and in Ishikawa cells compared to HeLa cells. The MTT and TUNEL assays results revealed the Ishikawa cells to be more sensitive to paclitaxel than HeLa cells and definite apoptosis occurred after a 24 h treatment. These results showed good agreement with the AFM results. Therefore, research on the morphological and biophysical changes by AFM in cancer cells will help to evaluate the anticancer activities of the drugs.     

The regulation of hypoxanthine guanine phosphoribosyl transferase activity through transfer of PRPP by metabolic cooperation

We have developed a method of relating changes in hypoxanthine guanine phosphoribosyl transferase (HGPRTase) activity to the rate of phosphoribosyl pyrophosphate (PRPP) synthesis in isolated cell lines and in co-cultures of different cell lines. Using this approach, we have determined the response of the HGPRTase activity of communication-competent and communication-incompetent cells to changes in PRPP content. The HGPRTase activity of HGPRT+ communication-competent NS cells responds to changes of their own PRPP level, as well as to changes of the PRPP level of HGPRT- cells with which they are co-cultured. In contrast, the HGPRTase activity of the HGPRT+, but communication-incompetent L929 cells responds to changes of their own PRPP content but not to changes of the PRPP content of the cocultured HGPRT- cells. These and other experiments show that PRPP is freely exchangeable between communication-competent cells and that the intracellular activity of HGPRTase in one cell can be regulated by changes in the levels of its substrate in another cell through metabolic cooperation. The results also indicate that HGPRTase normally functions at a small fraction of its total activity, and that this can be greatly increased by raising the intracellular PRPP levels. Furthermore, it is found that when communication-competent cells establish intercellular communication, they share a common pool of PRPP and of purine nucleotides. This approach can be used as the basis of a biochemical method for the quantitation of metabolic cooperation between cells.     

The effect of Ca on virus-cell fusion and permeability changes

Sendai virus-mediated permeability changes in Lettre cells or red blood cells are affected by extracellular Ca2+ in the following way: the lag period to onset of permeability changes is lengthened and the subsequent extent of leakage is reduced. Ca2+ neither stimulates nor inhibits fusion of the viral envelope to the plasma membrane of Lettre cells or red blood cells. It is concluded that Ca2+ protects cells against virally-induced permeability changes in a manner not involving membrane fusion.     

Phorbol esters and gene expression: the role of rapid changes in K+ transport in the induction of ornithine decarboxylase by 12-O- tetradecanoylphorbol-13-acetate in BALB/c 3T3 cells and a mutant cell line defective in Na+K+Cl- cotransport

A BALB/c 3T3 preadipose cell line defective in Na+K+Cl- cotransport (3T3-E12a cells) has been used to study the relationship between phorbol ester-induced rapid changes in cation fluxes and changes in expression of a gene known to be modulated by this agent. In contrast to its effect on parental 3T3 cells, 12-O-tetradecanoylphorbol-13-acetate (TPA) did not inhibit either furosemide-sensitive 86Rb+ influx or the rate of 86Rb+ efflux from preloaded mutant cells. TPA-induced changes in intracellular K+ content were diminished in 3T3-E12a cells as compared with parental cells. Thus, mutation of the Na+K+Cl- cotransport system renders overall potassium transport in mutant cells largely insensitive to modulation by TPA. The morphological and functional responses of 3T3 and 3T3-E12a cells to TPA were also compared. In contrast to the extensive and long-lasting changes in morphology of 3T3 cells after 0.16 microM TPA addition, only slight and shorter-lived morphological effects of TPA were observed in 3T3-E12a cells. The transport properties of mutant cells were not totally unresponsive to TPA since hexose transport (2-deoxyglucose uptake) could be stimulated in both cell types. To establish a possible link between early changes in cation fluxes and activation of gene expression by TPA, the induction of the enzyme ornithine decarboxylase (ODC) was studied in detail. Addition of fresh medium containing serum or exposure to hypoosmotic conditions resulted in the induction of ODC in both 3T3 and 3T3-E12a cells. However, TPA failed to cause an increase in ODC activity in mutant cells, although a substantial induction of the enzyme was seen in parental cells. These results suggest that rapid changes in ion fluxes mediated by the Na+K+Cl- cotransport system are necessary for at least one of the phorbol ester-induced changes in gene expression in responsive cells.     

Humidity Responses of Stomata and the Potassium Content of Guard Cells

Humidity responses of stomata and changes in the potassium contentof their guard cells were investigated in intact plants anddetached epidermal strips of Valerianella locusta (L.) Betcke.Potassium content was determined by Macallum‘s stain.It was found that changes in stomatal aperture caused by decreasingor increasing humidity were followed only after a delay by changesin the potassium content of the guard cells. By comparison,if stomatal movements occurred in response to changes in illuminationthe relative potassium content of the guard cells correlatedcontinuously with the changes in stomatal aperture. Since thepotassium content of the guard cells changed only after mostof the stomatal movements in response to changes in humiditywere completed changes in potassium content and humidity responsesof stomata can be described as following a hysteresis curve.     

Effects of temperature and cellular interactions on the mechanics and morphology of human cancer cells investigated by atomic force microscopy

Cell mechanics plays an important role in cellular physiological activities. Recent studies have shown that cellular mechanical properties are novel biomarkers for indicating the cell states. In this article, temperature-controllable atomic force microscopy(AFM) was applied to quantitatively investigate the effects of temperature and cellular interactions on the mechanics and morphology of human cancer cells. First, AFM indenting experiments were performed on six types of human cells to investigate the changes of cellular Young's modulus at different temperatures and the results showed that the mechanical responses to the changes of temperature were variable for different types of cancer cells. Second, AFM imaging experiments were performed to observe the morphological changes in living cells at different temperatures and the results showed the significant changes of cell morphology caused by the alterations of temperature. Finally, by co-culturing human cancer cells with human immune cells, the mechanical and morphological changes in cancer cells were investigated. The results showed that the co-culture of cancer cells and immune cells could cause the distinct mechanical changes in cancer cells, but no significant morphological differences were observed. The experimental results improved our understanding of the effects of temperature and cellular interactions on the mechanics and morphology of cancer cells.     

Changes in surface area of intact guard cells are correlated with membrane internalization

Guard cells must maintain the integrity of the plasma membrane as they undergo large, rapid changes in volume. It has been assumed that changes in volume are accompanied by changes in surface area, but mechanisms for regulating plasma membrane surface area have not been identified in intact guard cells, and the extent to which surface area of the guard cells changes with volume has never been determined. The alternative hypothesis-that surface area remains approximately constant because of changes in shape-has not been investigated. To address these questions, we determined surface area for intact guard cells of Vicia faba as they underwent changes in volume in response to changes in the external osmotic potential. We also estimated membrane internalization for these cells. Epidermal peels were subjected to external solutions of varying osmotic potential to shrink and swell the guard cells. A membrane-specific fluorescent dye was used to identify the plasma membrane, and confocal microscopy was used to acquire a series of optical paradermal sections of the guard cell pair at each osmotic potential. Solid digital objects representing the guard cells were created from the membrane outlines identified in these paradermal sections, and surface area, volume, and various linear dimensions were determined for these solid objects. Surface area decreased by as much as 40% when external osmotic potential was increased from 0 to 1.5 MPa, and surface area varied linearly with volume. Membrane internalization was approximated by determining the amount of the fluorescence in the cell's interior. This value was shown to increase approximately linearly with decreases in the cell's surface area. The changes in surface area, volume, and membrane internalization were reversible when the guard cells were returned to a buffer solution with an osmotic potential of approximately zero. The data show that intact guard cells undergo changes in surface area that are too large to be accommodated by plasma membrane stretching and shrinkage and suggest that membrane is reversibly internalized to maintain cell integrity.     

Nongranulated cells in the mouse adenohypophysis

Summary Follicular cells in the mouse adenohypophysis were studied electron microscopically. These elements appear to be very similar to the marginal cells that delineate both sides of the hypophyseal cleft.The mouse differs from most other species in that the follicular cells in the pars distalis and the marginal cells look completely inactive in young, intact animals. This makes the mouse exceptionally favorable for correlating morphological changes in the cells of both types with changes in the physiological state of the animal. Different treatments applied in the present investigation all induced morphological reactions in the follicular and/or marginal cells; these reactions were generally similar. Thus, morphological changes in the follicular or marginal cells should be considered as general phenomena accompanying many changes in the physiological state of the animal, rather than as a specific result of the treatment applied.In three experiments, the follicular and marginal cells were involved in the digestion of waste material from other cells. It is suggested that the morphological changes in the other experiments should also be interpreted as signs of such an activity.In the pars tuberalis of the young, intact mouse the follicular cells may show characteristics that in the pars distalis are found only under experimental conditions. Therefore, the follicular cells in this part of the hypophysis are probably in an active state.     

Ultrastructural changes produced in Ehrlich ascites carcinoma cells by ultraviolet-visible radiation in the presence of melanins

Irradiation of Ehrlich ascites carcinoma (EAC) cells in the presence of pheomelanin, i.e., red hair melanin (RHM), has been reported to produce extensive cell lysis. Irradiation in the presence of eumelanin, i.e., black hair melanin (BHM), or irradiation in the absence of either type of melanin did not produce this effect. We observed that RHM particles penetrated the cell membrane without apparent structural damage to the cell or the cell membrane. Irradiation of the cells in the absence of melanin did not produce any changes in the ultrastructure of the cells. Incubation of the cells in the dark in the presence of RHM produced only minor structural, mainly cytoplasmic changes. Irradiation of the cells in the presence of RHM produced extensive ultrastructural changes prior to complete cell lysis; these changes were more severe than the effects of incubation of the cells in the dark in the presence of RHM. When the cells incubated in the dark or irradiated in the presence of latex particles or either one of the eumelanins particles, viz. BHM or synthetic dopa melanin, these particles did not penetrate into the cells or produce any ultrastructural changes. These particles were in fact not even ingested by the cells.     

Rapid pH changes associated with synaptic transmission in isolated slices of the mammalian hippocampus

To perform rapid optical detection of possible pH changes accompanying electrical activity hippocampal slices were stained with pH indicator--phenol red (0.2 mM). Electrical response of granular and pyramidal cells was evoked by stimulation of perforant path, Schaffer collateral and commissural afferents in the stratum radiatum. Biphasic pH changes occurred both in pyramidal and granular cells: rapid acid changes, with the maximum reached in several msec, were followed by alkaline changes lasting up to one sec. pH changes disappeared with the blocking of synaptic transmission by Mg2+ (10 mM) and were absent in antidromic stimulation of granular cells. pH changes are believed to be related to the processes accompanying synaptic transmission.     

ULTRASTRUCTURAL CHANGES IN EMBRYONIC ECTODERMAL CELLS OF THE NEURULATING RAT EMBRYO

Embryonic ectodermal cells of rat embryos were examined by light and electron microscopy during the early stage of neurulation. Before the onset of neurulation (day 9–6 hr embryos), the cells underwent certain characteristic ultrastructural changes; that is, apical cytoplasmic protrusions and free spherules appeared, numerous vacuoles were formed in the cytoplasm, mitochondria showed ballooning, and the endoplasmic reticulum became dilated. The amniotic cells derived from the embryonic ectoderm exhibited the same ultrastructural changes, but those from the extraembryonic mesoderm did not. Embryonic mesodermal cells and neuroectodermal cells also did not show these changes. In the middle stage of neurulation (day 9–12 hr embryos), the embryonic ectodermal cells and the amniotic cells derived from the embryonic ectoderm assumed a flat squamous shape. None of the ultrastructural changes observed in day 9–6 hr embryos were noted in these cells. The functional significance of the production of apical cytoplasmic protrusions and free spherules in the embryonic ectodermal cells and amniotic cells is discussed in relation to similar phenomena reported to occur in other cell types.     

Early changes in the membrane of HeLa cells adsorbing Sendai virus under conditions of fusion.

Adsorption of Sendai virus at high multiplicity (500-1,000 HAU/10(6) cells) to HeLa cells grown in monolayers causes immediate changes in the ion barrier of the cell membrane, as well as changes in the morphology of the virus-treated cells. Within minutes of adsorption the cells begin to lose potassium and an extensive influx of ions into the cells occurs. Concomitantly with these changes, the cell membrane becomes depolarized, and the resting potential across its membrane decreases. Twenty to sixty minutes post adsorption the damage to the cell membrane is repaired, and both the potassium uptake and the resting potential return to their pre-exposure values. Scanning electron-micrographs of Sendai infected cells incubated at 37 degrees C show formation of bridging microvilli in a zipper-like fashion within two to five minutes post-adsorption; 30 to 60 minutes thereafter the majority of cells in the monolayer are fused. Biochemical changes induced by virus adsorption and the role of Ca++ ions in the observed effects are discussed.     

The relation between connective tissue cells and intercellular substances, including basement membranes.

Basement membranes are distributed widely in the body forming an extracellular matrix for epithelial and endothelial cells. The collagenous and glycoprotein constituents of basement membranes are synthesized by these two cell types. Disturbance of the interactions between basement membranes and their associated epithelial and endothelial cells can lead to the pathological changes seen in diseases involving basement membranes. These changes are illustrated here by reference to glomerulonephritis induced by the deposition of immune complexes in the glomerulus of the kidney, and chronic inflammatory changes occurring in the lung after inhalation of asbestos. In these diseases basement membrane changes can occur in several ways. Hydrolytic enzymes released from inflammatory cells degrade basement membranes while other constituents by epithelial and endothelial cells. Alternatively the physical separation of epithelial and endothelial cells from their basement membrances by space-occupying substances such as immune complexes can interfere with feedback mechanisms leading to synthesis of basement membrane constituents and cell proliferation. Studies of these pathological changes at a cellular level should shed new light on the ways in which cells interact with their pericellular environment.     

Tumourigenicity, cell-surface glycoprotein changes and ornithine decarboxylase gene pattern in Ehrlich ascites-carcinoma cells.

We selected a 2-difluoromethylornithine-resistant Ehrlich ascites-carcinoma cell line that grows in the presence of 20 mM-difluoromethylornithine. These cells contain 10-20 times the normal amount of hybridizable sequences for ornithine decarboxylase (EC 4.1.1.17) in their genomic DNA. We used these gene-amplified cells, their revertant counterparts (grown in the absence of the drug after an established gene amplification) and tumour cells grown in the presence of putrescine to investigate the changes of ornithine decarboxylase gene pattern and simultaneously occurring phenotypic changes, such as tumourigenicity and the expression of cell-surface glycoproteins. In the tumour cells reverted back to the normal gene frequency, not only did the amplified sequences disappear, but there were also signs of gene re-arrangements seen as a "gene jump', when a signal evidently moved to a heavier restriction fragment. Similar gene re-arrangement likewise occurred in cells exposed to putrescine. Although the wild-type tumour cells and the gene-amplified cells readily grew in the peritoneal cavity of mice, the revertant cells and the putrescine-treated cells had lost their tumourigenicity in mice. Gene-amplified tumour cells and the revertant cells showed distinct changes in their surface glycoprotein pattern in comparison with the parental cell line. These findings indicate that alterations of ornithine decarboxylase gene pattern/dosage may be associated with phenotypic changes possibly related to the tumourigenicity of these carcinoma cells.     

Biology of reproduction in the squirrel monkey (Saimiri sciureus): III. Ultrastructure of the endometrial glandular cell during estrogen and progesterone feeding

Electron microscopic study of the endometrial glandular epithelium of the squirrel monkey (Saimiri sciureus) revealed the following three types of cells (1) principal, (2) mucus, and (3) ciliated cells. We further observed that these cells undergo morphologic changes in response to estrogen and progesterone feeding. The morphologic changes observed in the principal and mucus cells represent phases in the transformation of relatively poorly differentiated cells into mature functioning cells. The mucus cells elicit an accelerated maturative and functional change. The morphologic changes of the ciliated cells were least obvious.Supported by Grant HDO-1952-03 from the United States Public Health Service.     

The estimation of similarity in characteristics of the post-mitotic daughter L-929 cells during their migration along the substrate

Using time-lapse microscopy, spreading of the post-mitotic daughter cells has been studied. The work was performed on non-synchronized cells of established L-929 cell line. The study was aimed to characterize the morphology of the cells as they move along the substrate and to determine whether the area of the migrating cells changes nonrandom. Two new parameters have been proposed for comparison of cell morphology: the identity indicator (II) and the synchronism indicator (SI). Time-dependent changes in the area in pairs of cells were measured to calculate these parameters. The first indicator shows the degree of coincidence between the absolute values of the area in the pair of the cells, whereas the second indicator shows synchronism of the changes in the cell areas and does not depend on their absolute values. The lower are the indicators, the higher is the similarity in the time-dependent changes in the areas of cell pairs studied. The indicators were shown to be approximately 1.5-fold lower for the pairs of the post-mitotic daughter cells than those for any other pair of the cells. The results indicate a nonrandom pattern of change in the morphology of the cells during their movement along the substrate.     

Behaviour of bovine spermatozoa in media of varying osmolality and tonicity

The response of bovine spermatozoa to media of different osmolalities and tonicities was studied by several methods. Packed cell volumes of cells in a dilute medium were greater than in a reference medium (similar to seminal plasma), and were less in a more concentrated medium, but the changes were not correlated quantitatively with the changed properties of the media. Specific effects on the configuration of the midpiece and tail were observed by phase-contrast microscopy. All these effects were elicited by changes in osmolality rather than tonicity. Electronic volume sensing and optical density measurements did not detect these changes in the cells. The morphological changes were not uniform in the cells of any one sample, some cells being unresponsive; in a small proportion of responsive cells the effects of raised osmolality appeared to be reversible.     

Estradiol influences the mechanical properties of human fetal osteoblasts through cytoskeletal changes

Estrogen is known to have a direct effect on bone forming osteoblasts and bone resorbing osteoclasts. The cellular and molecular effects of estrogen on osteoblasts and osteoblasts-like cells have been extensively studied. However, the effect of estrogen on the mechanical property of osteoblasts has not been studied yet. It is important since mechanical property of the mechanosensory osteoblasts could be pivotal to its functionality in bone remodeling. This is the first study aimed to assess the direct effect of estradiol on the apparent elastic modulus (E¹) and corresponding cytoskeletal changes of human fetal osteoblasts (hFOB 1.19). The cells were cultured in either medium alone or medium supplemented with β-estradiol and then subjected to Atomic Force Microscopy indentation (AFM) to determine E¹. The underlying changes in cytoskeleton were studied by staining the cells with TRITC-Phalloidin. Following estradiol treatment, the cells were also tested for proliferation, alkaline phosphatase activity and mineralization. With estradiol treatment, E¹ of osteoblasts significantly decreased by 43–46%. The confocal images showed that the changes in f-actin network observed in estradiol treated cells can give rise to the changes in the stiffness of the cells. Estradiol also increases the inherent alkaline phosphatase activity of the cells. Estradiol induced stiffness changes of osteoblasts were not associated with changes in the synthesized mineralized matrix of the cells. Thus, a decrease in osteoblast stiffness with estrogen treatment was demonstrated in this study, with positive links to cytoskeletal changes. The estradiol associated changes in osteoblast mechanical properties could bear implications for bone remodeling and its mechanical integrity.