Cancer cell necrosis by autoschizis: Synergism of antitumor activity of vitamin C: Vitamin K3 on human bladder carcinoma T24 cells

Scanning and transmission electron microscopy and fluorescence light microscopy were employed to characterize the cytotoxic effects of vitamin C (VitC), vitamin K₃ (VitK₃) or a VitC:VK₃ combination on a human bladder carcinoma cell line (T24) following 1-h and 2-h vitamin treatment. T24 cells exposed to VitC alone exhibited membranous damage (blebs and endoplasmic extrusions, elongated microvilli). VitK₃-treated cells displayed greater membrane damage and enucleation than those treated with VitC as well as cytoplasmic defects characteristic of cytoskeletal damage. VitC:VitK₃-treated cells showed exaggerated membrane damage and an enucleation process in which the perikarya separate from the main cytoplasmic cell body by self-excision. Self-excisions continued for perikarya which contained an intact nucleus surrounded by damaged organelles. After further excisions of cytoplasm, the nuclei exhibited nucleolar segregation and chromatin decondensation followed by nuclear karryorhexis and karyolysis. This process of cell death induced by oxidative stress was named autoschizis because it showed both apoptotic and necrotic morphologic characteristics.     

The switch mechanism of the cell death mode from apoptosis to necrosis in menadione-treated human osteosarcoma cell line 143B cells

Time-dependent changes in the cell death mode from apoptosis to necrosis were studied in cultured 143B cells treated with menadione, an anti-cancerous drug, excluding a possible involvement of "secondary necrosis." The population of apoptotic cells judged by FITC-Annexin V and propidium iodide (PI) double staining reached its maximum at 6 hours after 100 microM menadione treatment followed by an abrupt decrease thereafter, while that of necrotic cells continuously increased reaching 90% at 24 hours. Electron microscopically, cells attached to the culture dish at 6 hours after the treatment consisted of two different types of cells: cells with typical apoptotic features occupying the major population and those with condensed nuclei and swollen cytoplasm. Cells attached to the culture dish at 8 hours after the treatment consisted exclusively of those with condensed nuclei and swollen cytoplasm. Mitochondria in these cells showed various structural changes: those swollen to various degrees with deposition of flocculent densities, or those with highly condensed matrix. Distinct decreases both in intracellular levels of ATP and caspase-3-like activities and remarkable elevations of intracellular levels of superoxide, which were partly suppressed by NAD(P)H oxidase inhibitors, occurred at 6 hours after the treatment. These results may suggest that distinct increases of the intracellular level of superoxide derived from plasma membrane NAD(P)H oxidase besides that from mitochondria have triggered the transition of cell death mode from apoptosis to necrosis. Transition of highly condensed mitochondria to extremely swollen ones may reflect necrotic processes in menadione-treated cells. The present study strongly suggests that time-dependent study is essential using the electron microscopic technique to analyze detailed processes in the changes of the cell death mode.     

Effect of estradiol and progesterone on thyroid gland in pigs: a histochemical, stereological, and ultrastructural study

The cellular and subcellular organization of thyroid follicular cells in peripubertal (6-month-old) male pigs treated with estradiol-dipropionate (Oe) plus progesterone (Pr) in combination on the first postnatal day was studied. A stereological method was used for morphometric determinations of the relative volume densities of the follicular epithelium, colloid and interstitium, and for establishing the epithelial height and index of activation rate. Statistically significant differences of the examined parameters between the control and Oe+Pr -treated groups were determined by Student's t-test. The subcellular organization of thyroid follicular cells was studied using transmission electron microscopy (TEM). When compared with the control group, in the Oe+Pr -treated pigs, thyroid follicles possessed a noticeably higher follicular epithelium when compared with the control animals. The observed changes were quantified and comparison between the experimental groups showed that the height, volume density of follicular epithelium, and index of activation rate were significantly (P < 0.05) increased, whereas the relative volume density of the colloid was significantly (P < 0.05) decreased. At the subcellular level the thyroid follicular cells of Oe+Pr -treated pigs were characterized by increased number of polysomes and dense bodies and extensive endoplasmatic reticulum. It was concluded that a single neonatal treatment with female gonadal steroids exerted a prolonged effect on the pig's thyroid, characterized by increased biosynthesis and reabsorption of the colloid by the follicular cells.     

The Frictional Response of VC(100) Surfaces: Influence of 1-Octanol and 2,2,2-Trifluoroethanol Adsorption

In this report, we present ultrahigh vacuum (UHV) atomic-scale measurements of the frictional response of the VC(100) surface and the influence on friction through the adsorption of 1-octanol (CH₃(CH₂)₇OH) and 2,2,2-trifluoroethanol (CF₃CH₂OH). Atomic force microscopy (AFM) has been used to determine the changes in interfacial friction and adhesion, while scanning tunneling microscopy (STM) has revealed changes in surface morphology upon adsorption. X-ray photoelectron spectroscopy (XPS) has been utilized to determine the composition of the surface formed through the reaction of these adsorbates with VC. Adsorption of 1-octanol on the VC(100) surface at room temperature causes a 15% reduction in the friction measured between a clean VC surface and a silicon nitride AFM tip. STM images, combined with XPS results, reveal that 1-octanol does not completely cover the surface and that saturation occurs approximately at a 500L exposure. Adsorption of 2,2,2-trifluoroethanol on the VC(100) surface at room temperature produces a significant increase in friction while at the same time producing a decrease in adhesion. These contrasting results are interpreted in terms of differences in interfacial shear strength, chemical composition, and the molecular details of the adsorbed layer.     

Hypoxia induced apoptosis of rat gastric mucosal cells by activating autophagy through HIF-1α/TERT/mTORC1 pathway

The pathogenesis of high altitude-related gastric mucosal injury remains poorly understood, this study aimed to investigate the role of autophagy in hypoxia-induced apoptosis of rat gastric mucosal cells. Rats were randomized into four groups which were maintained at an altitude of 400 m (P) or received no treatment (H), autophagy inducer rapamycin (H+AI) or autophagy inhibitor 3-MA (H+AB) at an altitude of 4,300 m for 1, 7, 14 and 21 days, respectively, and the morphology, ultrastructure, autophagy, and apoptosis of gastric mucosal tissues were examined. Gastric mucosal epithelial cells CC-R039 were cultured under conditions of normoxia, 2% O2 (hypoxia), or 2% O2+anti-mTORC1 for 0, 24, 48, and 72 h, respectively, and the autophagy and apoptosis were analyzed. CC-R039 cells were transfected with siHIF-1α, siTERT, or siRNA and the autophagy was examined. The results showed that the exposure to hypoxia increased the autophagy and apoptosis of gastric mucosal cells in rats, and apoptosis was aggravated by rapamycin treatment but alleviated by 3-MA treatment. Increased duration of hypoxia from 0 to 72 h could increase the autophagy and apoptosis but decrease the proliferation of gastric mucosal cells. Inhibition of mTORC1 with rapamycin led to further increase in apoptosis and even substantial cell death, and inhibition of HIF- 1α and TERT increased mTORC1 expression and reduced autophagy. Moreover, the inhibition of HIF-1α reduced TERT expression. In conclusion, hypoxia could induce apoptosis of rat gastric mucosal cells by activating autophagy through HIF-1α/TERT/mTORC1 pathway     

An extract of <i>Hypericum perforatum</i> induces wound healing through inhibitions of Ca²⁺ mobilizations,mitochondrial oxidative stress and cell death in epithelial cells: Involvement of TRPM2 channels

The wound is induced by several mechanical and metabolic factors. In the etiology of the wound recovery, excessive oxidative stress, calcium ion (Ca²⁺) influx, and apoptosis have important roles. Ca²⁺-permeable TRPM2 channel is activated by oxidative stress. Protective roles of Hypericum perforatum extract (HP) on the mechanical nerve injury-induced apoptosis and oxidative toxicity through regulation of TRPM2 in the experimental animals were recently reported. The potential protective roles in HP treatment were evaluated on the TRPM2-mediated cellular oxidative toxicity in the renal epithelium (MPK) cells. The cells were divided into three groups as control, wound, and wound + HP treatment (75 µM for 72 h). Wound diameters were more importantly decreased in the wound+HP group than in the wound group. In addition, the results of laser confocal microscopy analyses indicated protective roles of HP and TRPM2 antagonists (N-(p-Amylcinnamoyl) anthranilic acid and 2-aminoethyl diphenylborinate) against the wound-induced increase of Ca²⁺ influx and mitochondrial ROS production. The wound-induced increase of early (annexin V-FITC) apoptosis and late (propidium iodide) apoptosis were also decreased in the cells by the HP treatment. In conclusion, HP treatment acted protective effects against wound-mediated oxidative cell toxicity and apoptosis through TRPM2 inhibition. These effects may be attributed to their potent antioxidant effect.     

Quantitative analysis of mast cell structure

Fixed and embedded normal rat peritoneal mast cells were studied by light and electron microscopy, utilizing stereological methods to obtain quantitative data on their structure. The diameters of the mast cells and their nuclei averaged 10.9 and 5.8 μm respectively. The volume of the individual mast cell granule was estimated to 0.3 μm³. About 53% of the cytoplasmic volume was occupied by granules, and 2% by mitochondria. 11% of the cell volume was taken up by the nucleus. The average number of granules per cell was calculated to about 1020. Quantitative biochemical data on mast cells, extrapolated from the literature and applied to the calculated figures above, yield the following results with respect to mast granule contents-heparin: 95 × 10^?3 pg, histamine: 30 × 10^?3 pg and 5-hydroxytryptamine: 1.3 × 10^?3 pg per mast cell granule.     

Resonance Rayleigh Scattering of K2Zn3[Fe(CN)6]2 Nanoparticles and its Application for the Determination of Vitamin C

Abstract

In Britton-Robinson buffer medium, (pH 4.43), potassium ferricyanide (K₃[Fe(CN)₆]) could react with vitamin C (VC) to produce potassium ferrocyanide (K₄[Fe(CN)₆]), which further reacted with Zn²⁺ to form potassium zinc hexacyanoferate K₂Zn₃[Fe(CN)₆]₂ nanoparticles. The shapes and diameters of the K₂Zn₃[Fe(CN)₆]₂ nanoparticles have been observed with transmission electron microscopy, which showed the shapes of these nanoparticles was cubic and their average sizes were about 50 nm in the presence of 2.0 × 10^?5 mol L^?1 VC. The characteristics of resonance Rayleigh scattering (RRS) spectra of this reaction have been studied. The optimum reaction condition for the determination of VC has been investigated. It was found that the RRS intensity of the system at the RRS peak of 363.4 nm was proportional to the VC concentration in the range of 4.0?80.0 µmol L^?1, and the detection limit (3σ) for VC was 0.075 µmol L^?1. A novel and simple RRS method for the determination of VC based on the formation of K₂Zn₃[Fe(CN)₆]₂ nanoparticles has been established.     

Reflection across plant cell boundaries in confocal laser scanning microscopy

The fluorescence patterns of proteins tagged with the green fluorescent protein (GFP) and its derivatives are routinely used in conjunction with confocal laser scanning microscopy to identify their sub-cellular localization in plant cells. GFP-tagged proteins localized to plasmodesmata, the intercellular junctions of plants, are often identified by single or paired punctate labelling across the cell wall. The observation of paired puncta, or 'doublets', across cell boundaries in tissues that have been transformed through biolistic bombardment is unexpected if there is no intercellular movement of the GFP-tagged protein, since bombardment usually leads to the transformation of single, isolated cells. We expressed a putative plasmodesmal protein tagged with GFP by bombarding Allium porrum epidermal cells and assessed the nature of the doublets observed at the cell boundaries. Doublets were formed when fluorescent spots were abutting a cell boundary and were only observable at certain focal planes. Fluorescence emitted from the half of a doublet lying outside the transformed cells was polarized. Optical simulations performed using finite-difference time-domain computations showed a dramatic distortion of the confocal microscope's point spread function when imaging voxels close to the plant cell wall due to refractive index differences between the wall and the cytosol. Consequently, axially and radially out-of-focus light could be detected. A model of this phenomenon suggests how a doublet may form when imaging only a single real fluorescent body in the vicinity of a plant cell wall using confocal microscopy. We suggest, therefore, that the appearance of doublets across cell boundaries is insufficient evidence for plasmodesmal localization due to the effects of the cell wall on the reflection and scattering of light.     

Comparative atomic force and scanning electron microscopy: an investigation on fenestrated endothelial cells in vitro

Rat liver sinusoidal endothelial cells (LEC) contain fenestrae, which are clustered in sieve plates. Fenestrae control the exchange of fluids, solutes and particles between the sinusoidal blood and the space of Disse, which at its back side is flanked by the microvillous surface of the parenchymal cells. The surface of LEC can optimally be imaged by scanning electron microscopy (SEM), and SEM images can be used to study dynamic changes in fenestrae by comparing fixed specimens subjected to different experimental conditions. Unfortunately, the SEM allows only investigation of fixed, dried and coated specimens. Recently, the use of atomic force microscopy (AFM) was introduced for analysing the cell surface, independent of complicated preparation techniques. We used the AFM for the investigation of cultured LEC surfaces and the study of morphological changes of fenestrae. SEM served as a conventional reference.
AFM images of LEC show structures that correlate well with SEM images. Dried-coated, dried-uncoated and wet-fixed LEC show a central bulging nucleus and flat fenestrated cellular processes. It was also possible to obtain height information which is not available in SEM. After treatment with ethanol or serotonin the diameters of fenestrae increased (+6%) and decreased (−15%), respectively. The same alterations of fenestrae could be distinguished by measuring AFM images of dried-coated, dried-uncoated and wet-fixed LEC. Comparison of dried-coated (SEM) and wet-fixed (AFM) fenestrae indicated a mean shrinkage of 20% in SEM preparations. In conclusion, high-resolution imaging with AFM of the cell surface of cultured LEC can be performed on dried-coated, dried-uncoated and wet-fixed LEC, which was hitherto only possible with fixed, dried and coated preparations in SEM and transmission electron microscopy (TEM).     

Divicine induces endothelial cells injury and its potential mechanism

Divicine is an active pyrimidine aglycone, generated from vicine by the enzyme β-glucosidase upon ingestion of fava beans. In this study, we investigated the effect of divicine on cultured human umbilical vein endothelial cells (HUVECs) and explored the potential mechanisms. Incubation HUVECs with 18.5–85.1 μM divicine resulted in a concentration and time dependent decrease of cell viability, followed by decrease of cellular reduced glutathione, as well as increase of reactive oxygen species (ROS), malondialdehyde (MDA), and labile iron pool. Transmission electron microscopy confirmed that the divicine treated HUVECs’ mitochondria had shrunk. Importantly, the administration of desferrioxamine, an iron chelator, to the divicine treated HUVECs significantly reduced iron overload and cell death and decreased cellular ROS and MDA. These results demonstrated that divicine could cause damaging of endothelial cells, and ferroptosis might be involved in divicine induced HUVECs injury, reminding long term ingestion of fava beans might be harmful to vascular system, especially for those suffering from glucose 6-phosphate dehydrogenase deficiency.     

Immunohistochemical study on the distribution of sodium-dependent vitamin C transporters in the respiratory system of adult rat

As vitamin C (L-ascorbic acid, VC) is known to be essential for many enzymatic reactions, the study on the transport mechanism of VC through cytoplasmic membrane is crucial to understanding physiological role of VC in cells and the respiratory system. In this regard, the study on the newly identified sodium-dependent VC transporters (SVCTs), SVCT1 and SVCT2, is required in organs that contain high concentration of VC. We have shown the distribution of SVCT proteins in the respiratory system, which has been reported to be one of the organs with a high concentration of VC, using immunohistochemical techniques. In the present study, intense SVCT immunoreactivities (IRs) were mainly localized in the respiratory system epithelial cells. In the trachea, both SVCT1 and 2 were localized in the psuedostratified ciliated columnar epithelium. In the terminal bronchiole, SVCT1 and 2 IRs were mainly observed in the apical portion of the simple columnar epithelium. In addition, SVCT IRs was localized within the cell membrane of some alveolar cells, even though we could not identify the exact cell types. These results provide the first evidence that intense SVCT1 and 2 IRs were found in the apical portion of the respiratory epithelial cells, suggesting that SVCT proteins in the apical portion could transport the reduced form of VC included in the airway surface liquid into the respiratory epithelial cells.     

Functionalized magnetic nanoparticles attenuate cancer cells proliferation: Transmission electron microscopy analysis

The penetration and transportation of nanoparticles (NPs) inside the cancer cells is critical to study. In this article, cancer cells (HCT‐116) were treated with functionalized magnetic NPs for the period of 48 hr and studied their ultrastructure by transmission electron microscopy (TEM). The NPs‐treated cells were prepared by chemical fixation and sliced into electron‐transparent arbitrary sections (200 × 200 μm²) by ultramicrotome. Major events of NPs–cell interaction, such as penetration of NPs, encapsulation of NPs into the intracellular compartments, transportation of NPs, and NPs exit, were examined by TEM to understand the mechanism of cell death. The NPs showed the uniform spherical shape with broad size distribution (100–400 nm), while cells displayed irregular morphology with average diameter ~5 μm. Our results showed the successful penetration of NPs deep into the cell, encapsulation, transportation, and exocytosis. Furthermore, we tested the different concentrations (0, 1.5, 12.5, and 50 μg/ml) of NPs on cancer cells and evaluated the cell viability. Laser confocal microscopy and colorimetric analysis together demonstrated that the cell viability is a dose‐dependent phenomenon, where 50 μg/ml specimen showed the highest killing of cancer cells compared to other dosages.     

AFM剖析纳米TiO2自清洁玻璃表面形貌

采用AFM对纳米TiO2自清洁玻璃表面形貌进行剖析、发现玻璃膜表面纳米TiO2粒径随TiO2溶胶浓度增大而增大，其在可见光条件下玻璃表面超亲水性随溶胶浓度提高而下降；稀土离子掺杂使纳米TiO2粒径变小，Ce^3＋掺杂使TiO2柱径从50nm变为30nm，玻璃的可见光透过率提高．从7了％提高到86％，对有机物的光催化效率提高、在90min内，对甲泰的分解率从90．02％提高到92．85％；钢化处理对纳米TiO2柱径的影响不大，但使镀膜玻璃的可见光透过率从86％提高到88％，Ce^3＋掺杂的钢化玻璃的TiO2对甲苯的分解率从92.85％提高到93．15％，分解时间从90min缩短到60min，Y^3＋掺杂对TiO2的影响小于Ce^3＋掺杂。     

Combined autoradiography and histochemistry: the simultaneous detection of 6-3H thymidine and acid phosphatase activity in cryostate sections.

A method is described for the simultaneous demonstration of 6-3H thymidine incorporation and acid phosphatase activity in cryostae sections of mouse thymus. The method enables a comparison of mitosis and acid hydrolase activity to be made in the same tissue section. In 8-week-old mice acid phosphatase positive cells reprsent 1.23 +/- 0.06% of the total population and 8.4 +/- 0.27% of the cells incorporate tritiated thymidine. Acid phosphatase activity can be used to estimate cell autolysis and death. The implication of the method in relation to tissue dynamics is discussed.     

Combining quantitative 2D and 3D image analysis in the serial block face SEM: application to secretory organelles of pancreatic islet cells

A combination of two‐dimensional (2D) and three‐dimensional (3D) analyses of tissue volume ultrastructure acquired by serial block face scanning electron microscopy can greatly shorten the time required to obtain quantitative information from big data sets that contain many billions of voxels. Thus, to analyse the number of organelles of a specific type, or the total volume enclosed by a population of organelles within a cell, it is possible to estimate the number density or volume fraction of that organelle using a stereological approach to analyse randomly selected 2D block face views through the cells, and to combine such estimates with precise measurement of 3D cell volumes by delineating the plasma membrane in successive block face images. The validity of such an approach can be easily tested since the entire 3D tissue volume is available in the serial block face scanning electron microscopy data set. We have applied this hybrid 3D/2D technique to determine the number of secretory granules in the endocrine α and β cells of mouse pancreatic islets of Langerhans, and have been able to estimate the total insulin content of a β cell.     

Scanning electron microscopy with an ionic liquid reveals the loss of mitotic protrusions of cells during the epithelial-mesenchymal transition

Epithelial-mesenchymal transition (EMT) is a key event in cancer metastasis and is characterized by increase in cell motility, increase in expression of mesenchymal cell markers, loss of proteins from cell-to-cell junction complexes, and changes in cell morphology. Here, the morphological effects of a representative EMT inducer, transforming growth factor (TGF)-β1, were investigated in human lung adenocarcinoma (A549) cells and pancreatic carcinoma (Panc-1) cells. TGF-β1 caused morphological changes characteristic of EMT, and immunostaining showed loss of E-cadherin from cell-to-cell junction complexes in addition to the upregulation of the mesenchymal marker vimentin. During scanning electron microscopy (SEM) with an ionic liquid, we observed EMT-specific morphological changes, including the formation of various cell protrusions. Interestingly, filopodia in mitotic cells were clearly observed by SEM, and the number of these filopodia in TFG-β1-treated mitotic cells was reduced significantly. We conclude that this reduction in such mitotic protrusions is a novel effect of TGF-β1 and may contribute to EMT.     

Investigation of integrin expression on the surface of osteoblast-like cells by atomic force microscopy

The transforming growth factor β1 (TGF-β1) is a human cytokine which has been demonstrated to modulate cell surface integrin repertoire. In this work integrin expression in response to TGF-β1 stimulation has been investigated on the surface of human osteoblast-like cells. We used atomic force microscopy (AFM) and confocal laser scanning microscopy to assess integrin expression and to evaluate their distribution over the dorsal side of the plasma membrane. AFM probes have been covalently functionalised with monoclonal antibodies specific to the β1 integrin subunit. Force curves have been collected in order to obtain maps of the interaction between the immobilized antibody and the respective cell membrane receptors. Adhesion peaks have been automatically detected by means of an ad hoc developed data analysis software. The specificity of the detected interactions has been assessed by adding free antibody in the solution and monitoring the dramatic decrease in the recorded interactions. In addition, the effect of TGF-β1 treatment on both the fluorescence signal and the adhesion events has been tested. The level of expression of the β1 integrin subunit was enhanced by TGF-β1. As a further analysis, the adhesion force of the single living cells to the substrate was measured by laterally pushing the cell with the AFM tip and measuring the force necessary to displace it. The treatment with TGF-β1 resulted in a decrease of the cell/substrate adhesion force. Results obtained by AFM have been validated by confocal laser scanning microscopy thus demonstrating the high potential of the AFM technique for the investigation of cell surface receptors distribution and trafficking at the nanoscale.