Apoptotic pathways depend on the target enzymatic activity and not on the triggering agent

Molt-4 human leukemia cells were triggered to apoptosis by various agents with different mechanisms of action. Staurosporine, a protein kinase C (PKC) inhibitor; camptothecin, a topoisomerase I blocking drug; and tiazofurin, an inhibitor of inosine 5'-phosphate dehydrogenase (IMPDH), were used. Ultrastructural analysis showed morphologic changes characteristic of apoptosis that were very similar for all three agents. Nevertheless, DNA oligonucleosomic fragmentation was not detectable by agarose gel electrophoresis. However, a genomic DNA cleavage appeared after pulse-field gel electrophoresis (PFGE) in cells treated with these agents for 24 h. Furthermore, in situ nick translation (NT) showed a finely spotted nuclear labelling in staurosporine-treated cells and a compact fluorescence after camptothecin incubation. In tiazofurin-treated cells an intermediate pattern was found. Therefore, apoptotic agents with different mechanisms of action induced the formation of large genomic DNA fragments and very similar ultrastructural changes. Therefore, both phenomena and the closely related apoptosis progression depend on target cell machinery and not on the triggering agent.     

Ultrastructural characterization of apoptotic granulosa cells in caprine ovary

The unique phenomenon of cell proliferation and apoptosis is encountered in the ovarian follicles undergoing early stages of atresia. The aim of this study was to verify the morphological variations in these two physiologically distinct processes operating in antral follicles of caprine ovaries using histological and ultrastructural techniques. Histologically the degenerating granulosa cells were characterized by condensed cytoplasm, and nucleus fragmentation in hazy cytosol. The pyknotic nuclei of degenerating cells stained darkly with haematoxylin and giemsa while the cytoplasm was eosinophilic. Under electron microscopy, apoptosis was marked by asymmetrical shrinkage, vacuolization of cytoplasm, swollen and vacuolated mitochondria, increased irregularity and/or fragmentation of nucleus, chromatin condensation and finally, production of membrane enclosed nuclear fragments containing intracellular material, the apoptotic bodies. The parallel use of these two methods on caprine ovaries has enabled us to analyse the decline in the frequency of granulosa cells during follicular atresia due to apoptosis.     

Melatonin prevents mitochondrial dysfunctions and death in differentiated skeletal muscle cells

Oxidative stress increase induces cellular damage and apoptosis activation, a mechanism believed to represent a final common pathway correlated to sarcopenia and many skeletal muscle disorders. The goal of this study is to evaluate if melatonin, a ROS scavenger molecule, is able to counteract or modulate myotube death. Here, differentiated C2C12 skeletal muscle cells have been treated with melatonin before chemicals known to induce apoptotic death and oxidative stress, and its effect has been investigated by means of morpho‐functional analyses. Ultrastructural observations show melatonin protection against triggers by the reducing of membrane blebbing, chromatin condensation, myonuclei loss and in situ DNA cleavage. Moreover, melatonin is able to prevent mitochondrial dysfunctions which occur in myotubes exposed to the trigger alone. These findings demonstrate melatonin ability in preventing apoptotic cell death in skeletal muscle fibers in vitro, suggesting for this molecule a potential therapeutic role in the treatment of various muscle disorders.     

Apoptogenic effect of the lipophilic <i>o</i>-naphthoquinone CG 10-248 on rat hepatocytes: light and electron microscopy studies

CG 10-248 (3,4-dihydro-2,2-dimethyl-9-chloro-2H-naphtho[1,2b]pyran- 5,6-dione; CG-NQ), a β-lapachone analogue, modified the ultrastructure of rat hepatocytes, as demonstrated by light and electron microscopy. After 4 h incubation with 100 µM CG-NQ, the following effects were observed: (a) nuclear chromatin condensation; (b) chromatin fragmentation; (c) displacement of mitochondria, concentrated around the nucleus; (d) disruption or expansion of mitochondrial outer or inner membranes, respectively; (e) displacement and alteration of endoplasmic reticulum (rough and smooth); (f) decrease of microvilli; (g) blebbing of plasma membrane and production of apoptotic bodies formed by folding of plasma membrane fragments around mitochondria or peroxysomes; and (h) production of hydrogen peroxide. Expression of such effects varied according to hepatocyte samples and taken together strongly support an apoptotic action of CG-NQ dependent on “reactive oxygen species”.     

Cardioprotective potential of curcumin against norepinephrine‐induced cell death: a microscopic study

Cardiomyopathy and associated heart failure continues to be one of the most severe complications that threaten a large population. Curcumin, one of the three curcuminoids of the spice turmeric, is very well known for a multitude of health benefits and functions. Norepinephrine (NE), a catecholamine and also a stress hormone may cause the cardiomyocytes to develop increased sensitivity to death with its increasing concentrations. In this study, we investigated the cardioprotective effect of curcumin in NE‐induced cardiac apoptosis using several fluorescent and nonfluorescent microscopic techniques like DAPI, PI, Giemsa, PicroSirius and TUNEL. The aim of the study was to assess the effect of curcumin in preventing the occurrence of features underlying apoptosis such as nuclear disruption, chromatin condensation, DNA fragmentation and alterations in mitochondrial membrane permeability. Our results show that curcumin protects the cardiomyocytes against apoptosis significantly and also helps them to revert to their normal physiological state. Hence, we propose that curcumin has the potential to act as a therapeutic agent for the attenuation of NE‐induced cardiac cell death and modulation of apoptosis in H9c2 cardiomyocytes.     

In vitro antitumor activity of broccolini seeds extracts

Broccolini (Brassica oleracea Italica × Alboglabra) is a hybrid of broccoli and kai‐lan, Chinese broccoli. To date, no report on antitumor activity of Broccolini (NOT Broccoli) is available. In this study, we evaluated the antiproliferative effects of broccolini seeds extract (BSE) on human lung and ovarian cancer cells. It was found that BSE induces A549 and OVCAR‐3 cells apoptosis in a dose‐dependent manner by using MTT assay. The IC₅₀ values of BSE in A549 and OVCAR‐3 cells were estimated to be 81.94 and 78.6 µg/ml, respectively. Furthermore, the phase contrast microscope showed that in high‐dose group (90～120 µg/ml), the morphology structure of OVCAR‐3 cells become irregular and exhibited characteristics of apoptosis such as cell membrane shrinkage, condensation and fragmentation of nuclear chromatin as well as formation of apoptotic bodies. SCANNING 33:402–404, 2011. © 2011 Wiley Periodicals, Inc.     

Self‐assembled monolayers of DNA on cysteamine modified Au(111) surface: Atomic force microscopy study

The λ‐DNA molecules self‐assemble on cysteamine‐modified gold (111) surface to form flat‐lying self‐assembled monolayers (SAMs). The formation kinetics of such DNA SAMs is studied by atomic force microscopy (AFM). AFM results show that DNA molecules do not arrange themselves on cysteamine‐modified gold (111) surface into a well‐ordered monolayer. It is also found that the surface density of DNA monolayer does not increase as the DNA concentration increases. The high temperature of DNA solution and the immersing in ultrapure water produce some obvious DNA bundles. Whereas divalent cations in DNA solution result in the formation of more compact DNA films. The obtained information may be useful for practical application of the DNA films and further theoretical studies. Microsc. Res. Tech., 2009. © 2009 Wiley‐Liss, Inc.     

Agmatine pretreatment protects retinal ganglion cells (RGC-5 cell line) from oxidative stress <i>in vitro</i>

Agmatine, 2-(4-aminobutyl)guanidine, has been reported to have neuroprotective effects against various neuronal damages. In this study it was investigated whether agmatine pretreatment rescues the retinal ganglion cells from oxidative injury in vitro. After differentiation of transformed rat retinal ganglion cells (RGC-5 cell line) with staurosporine, agmatine (0.0 to 100.0 μM) pretreatment was performed for 2 hours. Subsequently, they were exposed to hydrogen peroxide (0.0 to 2.5 mM) as an oxidative stress. Cell viability was monitored for up to 48 hours with the lactate dehydrogenase (LDH) assay and apoptosis was examined by the terminal deoxynucleotide transferase-mediated terminal uridine deoxynucleotidyl transferase nick end-labeling (TUNEL) method. As a result, differentiated RGC-5 cells were found to have decreased viability after addition of hydrogen peroxide in a dose-dependent manner. This hydrogen peroxide induced cytotoxicity caused apoptosis characterized by DNA fragmentation. Agmatine pretreatment not only increased cell viability but also attenuated DNA fragmentation. In conclusion, agmatine pretreatment demonstrated neuroprotective effects against oxidative stress induced by hydrogen peroxide in differentiated RGC-5 cells in vitro. This suggests a novel therapeutic strategy rescuing retinal ganglion cells from death caused by oxidative injury     

Nuclear pores in luteal cells during pregnancy and after parturition and pup removal in the rat. A freeze-fracture study

In a previous paper we described a pronounced increase of apoptotic nuclei in rat corpus luteum of pregnancy whose programmed chromatin degeneration was induced by the progesterone antagonist mifepristone. Those observations encouraged us to study the apoptotic nuclear membrane during pregnancy and after parturition and pup removal, by using a freeze-fracture technique which allows us to observe ‘en face’ the nuclear envelop and also permits nuclear pore counting. This study was complemented with the TUNEL assay (TdT-mediated dUTP nick-end labelling). Changes in nuclear pores during pregnancy begin with an intense reduction in number but still showing an even distribution on the nuclear membrane, never forming aggregations sharply separated from pore-free areas, which are characteristic of other apoptotic models. Electron microscopy of thin-sections shows, coincidently with findings in the freeze-fracture replicas, a moderately irregular aggregation of marginal heterochromatin condensations. After nuclear fragmentation and micronuclear formation, pores behave in the usual manner in other apoptotic models, i.e., mainly showing migrations of nuclear pores toward the chromatin-free areas. The present results support the hypothesis that nuclear pore complexes are dynamic structures, which permit their migration toward nuclear membrane areas devoid of chromatin aggregations that might block the nucleocytoplasmic transport in such areas.     

Effects of metals cadmium and chromium alone and in combination on the liver and kidney tissue of male Spraque‐Dawley rats: An ultrastructural and electron‐energy‐loss spectroscopy investigation

Heavy metal pollution has increased in the last decades. Water sources are contaminated and human exposure is often long term exposure to variable amounts of different metals. In this study, male Sprague‐Dawley rats were exposed via oral gavage for 28 days to cadmium (Cd) and chromium (Cr), alone and in combination at concentrations 1000 times the human World Health Organization's acceptable water limits. Rat equivalent dosages were used. Blood markers of liver and kidney function were measured, changes to cellular morphology was determined with transmission electron microscopy and the intracellular metal localisation was determined with the electron energy‐loss spectroscopy and energy filtered transmission electron microscopy analysis. Both Cd and Cr caused changes to the nuclear and mitochondrial membranes and irregular chromatin condensation of hepatocytes. Cr exposure caused dilation of the rough endoplasmic reticulum (rER). The combination caused nuclear and mitochondrial membrane damage as well as irregular chromatin condensation. In the kidney tissue, Cd caused irregular chromatin condensation in the cells of the proximal convoluted tubule (PCT). Cr caused changes to the outer nuclear and mitochondrial membrane and chromatin structure. The combination group caused membrane damage, irregular chromatin condensation and rER changes in the PCT. All the metal groups showed damage to the endothelial cells and pedicles, but not to the mesangial cells. Cd and Cr bio‐accumulation was observed in the nucleus, mitochondria and rER of the liver and kidney and therefore are responsible for the cellular observed damage that can cause functional changes to the tissues and organs.     

Technical Note : Prolonged exposure of human embryonic stem cells to heat shock induces necrotic cell death

We investigated the effects of prolonged heat shock treatment on human embryonic stem cell (hESC) viability. The hESC viability steadily declined with longer exposure to heat shock treatment (43ºC). After 4 h of exposure to heat shock at 43ºC, only 56.2 ± 1.5% of cells were viable. Viability subsequently declined to 37.0 ± 3.3% and 3.5 ± 0.7% after 8 h and 16 h, respectively of heat shock treatment at 43ºC. Transmission electron micrographs showed that the morphology of the dead/dying cells after heat shock treatment was characteristic of cellular necrosis with an uncondensed chromatin and a non-intact plasma membrane. This was further confirmed by flow cytometry analysis which showed that the DNA of the dead/ dying cells was still mostly intact, unlike the characteristic DNA fragmentation observed with apoptotic cells. In conclusion, prolonged exposure to heat shock treatment was detrimental to hESC viability. Hence, any future protocols developed for either the heat shock pre-conditioning of hESC prior to transplantation or for the temporary expression of specific genes with heat shock-responsive promoters should take these results into account; to achieve an optimal balance between the duration of heat shock exposure and the attainment of the desired effects.     

Key morphologic changes and DNA strand breaks in human lymphoid cells: Discriminating apoptosis from necrosis

Apoptosis is an important form of physiologic cell death displayed by an enormous variety of tissues under divergent conditions. The recent attention toward apoptosis in virtually all aspects of modern biology indicates that rapid and accurate differentiation between apoptosis and necrotic death is of considerable interest. Apoptosis is distinguishable from necrosis on the basis of several criteria. In this study, we undertook to examine the effects of mild hyperthermia (43°C leading to apoptotic death) and severe hyperthermia (50°C leading to necrotic killing) on associated DNA fragmentation. Using laser scanning and fluorescent microscopic evaluation of DNA “comets” in the single cell gel assay, we compared necrotic and apoptotic DNA damage in a variety of human leukemia and lymphoma cell lines at the level of the individual cell. We show that necrotic cells do display detectable DNA damage. We confirm our preliminary report that comet “tail moment” is sufficient to distinguish between necrotic and apoptotic DNA damage, while comet tail length may confuse the two forms. We report that a recovery period is necessary for expression of increasing apoptotic but not necrotic DNA damage. We show that apoptosis increases with prolonged hyperthermia and confirm that the mode of death changes from apoptosis to necrosis with higher heat loads, producing a greater fraction of cells showing damage. In addition, we show that for necrotic cells, DNA tail moment reflects sensitivity to prolonged exposure without a concomitant change in tail length.     

Endopolygalacturonase-gold complex: Parameters influencing the labelling of pectic acid in the cell walls of flax hypocotyl

An enzyme/colloidal-gold complex was prepared using a purified endopolygalacturonase (endoPGH, E.C. 3.2.1.15). Gold particles with a radius of 4·8 nm were prepared. To stabilize the gold particles, it was necessary to add a minimal amount of enzyme (approximately 0·5 μg) at a pH higher than its isoelectric pH. Under these conditions, the number of protein molecules adsorbed per gold particle was approximately one. The parameters influencing the diffusion of the endoPGH-gold complex to its target molecules were screened and tested through quantitative approaches: (i) the labelling efficiency q was calculated according to the equation for the diffusion of gold markers from the bulk solutions to target molecules and (ii) the labelling density N/S was measured by counting gold particles on electron micrographs. The highest value of N/S was obtained by determining the optimal values for the parameters outlined in the equation for q. To obtain the highest value of N/S, it was not necessary to choose values for these parameters which gave high values for q. The specificity of the probe towards homogalacturonic acid blocks was established through controls. The localization of these blocks was illustrated throughout the walls of different kinds of flax seeding cells.     

Immunoelectron microscopical distribution of histones H2B and H3 and protamines in the course of mouse spermiogenesis.

We have followed the fine structural distribution of two nucleosomal core histones, H2B and H3, and of protamines in the course of mouse spermiogenesis by means of specific antibodies and ultrastructural immunocytochemistry. Our results demonstrate that the nuclear labeling density of histone H2B decreases during steps 6-8 and then increases again in step 9-10 spermatids, while the labeling for histone H3 is constant throughout this period. In step 12 spermatids, the anti-H2B antibody labels mainly the central area of the nucleus. The first signs of protamine labeling are present in step 12 spermatids, where the gold grains can be found over the periphery of the nucleus. Later on, protamine labeling constantly increases and, by the end of spermiogenesis, the whole nucleus is labeled. We suggest that the morphological and structural differences between the central area and the periphery of mouse spermatids are, at least partly, due to a difference in the protein moiety associated with DNA. The central area, which is peculiar to the mouse and has been previously considered as a focus of chromatin condensation, represents, however, the last nuclear region containing histones and consequently the last area where the substitution of histones by protamines takes place.     

Morphologic and temporal analysis of vascular smooth muscle cell apoptosis induced by c-Myc and E1A

Apoptosis is a physiologic form of cell death present in many disease conditions. When the balance of mitosis versus apoptosis is altered, tumor-like growth or degeneration of tissues may ensue. This appears to occur in several diseases, including those of the cardiovascular system, where apoptosis plays a key role in atherosclerosis and restenosis following angioplasty. Since c-myc is upregulated in the pathogenesis of these diseases, we chose to study the sequential morphologic features of programmed cell death in vascular smooth muscle cells induced by c-myc and by the adenovirus early gene E1A. Morphology and timed events in apoptotic cell cultures were analyzed by scanning electron microscopy, transmission electron microscopy, and time-lapse videomicroscopy. We observed that both c-myc-and E1A-induced apoptosis (in serum-free medium) resulted in numerous, tightly packed clusters of apoptotic blebs, as well as in one or two asymmetrically larger blebs. Transmission electron microscopy analysis revealed the larger blebs contained mostly nuclear chromatin, whereas the many smaller fragments often had little or no chromatin. Time-lapse studies showed that apoptosis was induced at a slower rate in cells stably transfected with c-myc versus those stably transfected with E1A. The early changes of apoptosis, including cell shrinkage and intense blebbing, occurred in under 5 min in both cells. Slight alterations such as cell size and further rounding occurred up to 8 h following the initial changes of apoptosis. Rather than being a part of the apoptotic response, release from the culture floor almost entirely resulted from movement of the culture flask. These studies provide a framework of timed morphologic events for future mechanistic investigation into the key aspects of myc-and E1A-induced apoptosis in vascular smooth muscle.     

Segmentation of digitized histological sections for quantification of the muscularized vasculature in the mouse hind limb

Immunohistochemical tissue staining enhances microvasculature characteristics, including the smooth muscle in the medial layer of the vessel walls that is responsible for regulation of blood flow. The vasculature can be imaged in a comprehensive fashion using whole‐slide scanning. However, since each such image potentially contains hundreds of small vessels, manual vessel delineation and quantification is not practically feasible. In this work, we present a fully automatic segmentation and vasculature quantification algorithm for whole‐slide images. We evaluated its performance on tissue samples drawn from the hind limbs of wild‐type mice, stained for smooth muscle using 3,3'‐Diaminobenzidine (DAB) immunostain. The algorithm was designed to be robust to vessel fragmentation due to staining irregularity, and artefactual staining of nonvessel objects. Colour deconvolution was used to isolate the DAB stain for detection of vessel wall fragments. Complete vessels were reconstructed from the fragments by joining endpoints of topological skeletons. Automatic measures of vessel density, perimeter, wall area and local wall thickness were taken. The segmentation algorithm was validated against manual measures, resulting in a Dice similarity coefficient of 89%. The relationships observed between these measures were as expected from a biological standpoint, providing further reinforcement of the accuracy of this system. This system provides a fully automated and accurate means of measuring the arteriolar and venular morphology of vascular smooth muscle.     

Karyotyping of barley chromosomes by a new fluorescence banding technique combined with scanning probe microscopy

Fluorescence banding has been used to classify chromosomes, except those of barley. Four of the seven barley chromosomes are indistinguishable by length or arm ratio. C-banding has been used for classification; however, it requires a long aging period. Here, we describe a new fluorescence banding method for barley. The chromosomes are treated with warm acetate followed by staining with a fluorescent dye, YOYO-1. Using this method, all seven barley chromosomes can be clearly distinguished. Atomic force microscopy and scanning near-field microscopy analyses revealed that the surfaces of the banded chromosomes were flat, indicating that the fluorescence intensity reflected the internal DNA density or condensation of chromatin.     

Cell‐death assessment by fluorescent and nonfluorescent cytosolic and nuclear staining techniques

Apoptosis, a genetically programmed cellular event leads to biochemical and morphological changes in cells. Alterations in DNA caused by several factors affect nucleus and ultimately the entire cell leading to compromised function of the organ and organism. DNA, a master regulator of the cellular events, is an important biomolecule with regards to cell growth, cell death, cell migration and cell differentiation. It is therefore imperative to develop the staining techniques that may lead to visualize the changes in nucleus where DNA is housed, to comprehend the cellular pathophysiology. Over the years a number of nuclear staining techniques such as propidium iodide, Hoechst‐33342, 4’, 6‐diamidino‐2‐phenylindole (DAPI), Acridine orange–Ethidium bromide staining, among others have been developed to assess the changes in DNA. Some nonnuclear staining techniques such as Annexin‐V staining, which although does not stain DNA, but helps to identify the events that result from DNA alteration and leads to initiation of apoptotic cell death. In this review, we have briefly discussed some of the most commonly used fluorescent and nonfluorescent staining techniques that identify apoptotic changes in cell, DNA and the nucleus. These techniques help in differentiating several cellular and nuclear phenotypes that result from DNA damage and have been identified as specific to necrosis or early and late apoptosis as well as scores of other nuclear deformities occurring inside the cells.     

Regenerating titanium ventricular assist device surfaces after gold/palladium coating for scanning electron microscopy

Titanium is one of the most commonly used materials for implantable devices in humans. Scanning electron microscopy (SEM) serves as an important tool for imaging titanium surfaces and analyzing cells and other organic matter adhering to titanium implants. However, high‐vacuum SEM imaging of a nonconductive sample requires a conductive coating on the surface. A gold/palladium coating is commonly used and to date no method has been described to “clean” such gold/palladium covered surfaces for repeated experiments without etching the titanium itself. This constitutes a major problem with titanium‐based implantable devices which are very expensive and thus in short supply. Our objective was to devise a protocol to regenerate titaniumsurfaces after SEM analysis. In a series of experiments, titanium samples from implantable cardiac assist devices were coated with fibronectin, seeded with cells and then coated with gold/palladium for SEM analysis. X‐ray photoelectron spectroscopy spectra were obtained before and after five different cleaning protocols. Treatment with aqua regia (a 1:3 solution of concentrated nitric and hydrochloric acid), with or without ozonolysis, followed by sonication in soap solution and sonication in deionized water, allowed regenerating titanium surfaces to their original state. Atomic force microscopy confirmed that the established protocol did not alter the titanium microstructure. The protocol described herein is applicable to almost all titanium surfaces used in biomedical sciences and because of its short exposure time to aqua regia, will likely work for many titanium alloys as well. Microsc. Res. Tech., 2009. © 2009 Wiley‐Liss, Inc.     

Visualization of cellular DNA crosslinks by atomic force microscopy

Cellular DNA crosslinks are a type of DNA damage induced by toxic chemicals or high‐energy radiation. If damaged DNA is not rapidly repaired, cells will die or mutate. To evaluate the types of DNA damage and their influence on vital cell activities, it is necessary to be able to detect DNA crosslinks. To date, indirect methods such as alkaline elution, potassium chloride–sodium dodecyl sulfate assay and comet assay have been used to detect DNA damage. Direct morphological observation, on the other hand, may be a useful tool to differentiate the types of DNA damage. In this report, atomic force microscopy (AFM) has been employed to visualize the breakage and crosslinking of cellular DNA strands in cells treated with formaldehyde and hydrogen peroxide. Our results showed that toxic chemical‐induced crosslinking of cellular DNA occurred in a dose‐dependent manner. DNA conglomerates were observed with high concentrations of formaldehyde, and the AFM observations were consistent with those of a comet assay. Our experiments demonstrate that AFM is an efficient method to differentiate the types of DNA damage. SCANNING 31: 75–82, 2009. © 2009 Wiley Periodicals, Inc.