Proteome analysis of apoptotic cells

Apoptosis, a genetically determined form of cell death, is a central and complex process involved in the development of multicellular organisms in the maintenance of cell homeostasis. During apoptosis, a large number of proteins involved in transducing signals are posttranslationally modified. Classical proteomics, the combination of protein separation by two-dimensional gel electrophoresis (2DGE) and protein identification by mass spectrometry (MS), enabled the discovery of more than 100 proteins altered during apoptosis. Functional data about protein degradation, modification, translocation, and synthesis were obtained. In addition to classical proteomics, some specifically designed proteome studies were carried out to analyze specific apoptotic components such as the mitochondrial releasing factors, death-inducing signaling complex (DISC), inhibitor of apoptosis (IAP) interacting proteins, and caspases. The identification of main regulators significantly influenced the elucidation of the concept underlying apoptosis signaling. Thus, the application of detailed protein analytical methods in the young field of apoptosis research was particularly fruitful.     

Chronic stress effects on the apoptotic index of the adrenal cortex of pregnant rats.

Chronic stress by immobilization during gestation can alter several mechanisms that maintain homeostasis in adrenal gland. The aim of this work was to quantify the apoptotic index of adrenal cortex during mid-pregnancy and to prove cytological characteristics by electron microscopy. The apoptotic index did not present significant differences between the adrenal cortex areas of control and experimental rats in any of the three ages studied. The day of gestation influenced significantly on the apoptotic index in both groups. This index increased as gestation progressed. It may be concluded that chronic stress by immobilization might induce the increase of apoptotic index in adrenal cortex as gestation progresses which might be related variations of plasmatic corticosterone and prolactin, and to the decrease of specific growth factors. On the other hand, it might be concluded that each zone of adrenal cortex behaves independently in regards to apoptosis and cellular proliferation via paracrine and/or autocrine regulatory mechanisms without being affected by other zones.     

Major pathways for used oil disposal and recycling. Part 1

One of the main concerns with lubricating oil relates to used oil management for both industrial and engine oils, although the environmental impact of gasoline and diesel engine oils is the most critical. Provided that efficient management systems are in place, most used oil should not reach the environment, so, the major question is how to dispose of collected used oil. The first option lies in burning it as a fuel, the second in recycling (reclaiming, reprocessing, re‐refining). The latter allows recovery of mineral base oils, which are valuable constituents of crude oil. Mobile (on site) and fixed plants for industrial oil recycling will first be discussed, and the paper will look at the most modern re‐refining processes that produce base oils of as high quality as virgin base oils. Based on current re‐refining experience, the quality of finished lubricants blended from re‐refined base stocks is also noted. Re‐refining today may be of significant benefit to the economy and can, of course, protect the environment. All modern re‐refining technologies produce small amounts of by‐products in which toxic materials may have been concentrated. A final aspect of reprocessing used oil is to integrate it, after hydrogen treatment, into existing refineries. This valuable raw material can then be directly routed to a lube oil unit or even to a cracking unit for conversion to gasoline. The integration of used oil treatment processes into selected refineries may be the most effective pathway to used oil disposal. In this first part, the author looks at the nature of the problems associated with used oil, its use as a fuel, and simple recycling. He then goes on to look at major re‐refining processes, starting with hydrogenation (KTI, Mohawk, BERC/NIPER, and PROP technologies). Part 2 will describe other processes, including a range of vacuum distillation/clay treatment technologies.     

Major pathways for used oil disposal and recycling. Part 2

One of the main concerns with lubricating oil relates to used oil management for both industrial and engine oils, although the environmental impact of gasoline and diesel engine oils is the most critical. Provided that efficient management systems are in place, most used oil should not reach the environment, so, the major question is ‘how should we dispose of collected used oil?’ The first option lies in burning it as a fuel, the second in recycling (re‐claiming, reprocessing, re‐refining). The latter allows recovery of mineral base oils, which are valuable constituents of crude oil. In the first part of this paper, the author looked at the problems associated with used oil, its use as a fuel, and simple recycling. He went on to look at major re‐refining processes, starting with hydrogenation (KTI, Mohawk, BERC/NIPER, and PROP technologies). In Part 2 he covers other processes, including Safety Kleen, IFP/Snamprogetti, UOP Hylube, and vacuum distillation and clay treatment technologies.     

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.     

Fragmentation pathways of polymer ions

Tandem mass spectrometry (MS/MS) is increasingly applied to synthetic polymers to characterize chain‐end or in‐chain substituents, distinguish isobaric and isomeric species, and determine macromolecular connectivities and architectures. For confident structural assignments, the fragmentation mechanisms of polymer ions must be understood, as they provide guidelines on how to deduce the desired information from the fragments observed in MS/MS spectra. This article reviews the fragmentation pathways of synthetic polymer ions that have been energized to decompose via collisionally activated dissociation (CAD), the most widely used activation method in polymer analysis. The compounds discussed encompass polystyrenes, poly(2‐vinyl pyridine), polyacrylates, poly(vinyl acetate), aliphatic polyester copolymers, polyethers, and poly(dimethylsiloxane). For a number of these polymers, several substitution patterns and architectures are considered, and questions regarding the ionization agent and internal energy of the dissociating precursor ions are also addressed. Competing and consecutive dissociations are evaluated in terms of the structural insight they provide about the macromolecular structure. The fragmentation pathways of the diverse array of polymer ions examined fall into three categories, viz. (1) charge‐directed fragmentations, (2) charge‐remote rearrangements, and (3) charge‐remote fragmentations via radical intermediates. Charge‐remote processes predominate. Depending on the ionizing agent and the functional groups in the polymer, the incipient fragments arising by pathways (1)–(3) may form ion–molecule complexes that survive long enough to permit inter‐fragment hydrogen atom, proton, or hydride transfers. © 2010 Wiley Periodicals, Inc., Mass Spec Rev 30:523–559, 2011     

Fragmentation pathways of protonated peptides

The fragmentation pathways of protonated peptides are reviewed in the present paper paying special attention to classification of the known fragmentation channels into a simple hierarchy defined according to the chemistry involved. It is shown that the 'mobile proton' model of peptide fragmentation can be used to understand the MS/MS spectra of protonated peptides only in a qualitative manner rationalizing differences observed for low-energy collision induced dissociation of peptide ions having or lacking a mobile proton. To overcome this limitation, a deeper understanding of the dissociation chemistry of protonated peptides is needed. To this end use of the 'pathways in competition' (PIC) model that involves a detailed energetic and kinetic characterization of the major peptide fragmentation pathways (PFPs) is proposed. The known PFPs are described in detail including all the pre-dissociation, dissociation, and post-dissociation events. It is our hope that studies to further extend PIC will lead to semi-quantative understanding of the MS/MS spectra of protonated peptides which could be used to develop refined bioinformatics algorithms for MS/MS based proteomics. Experimental and computational data on the fragmentation of protonated peptides are reevaluated from the point of view of the PIC model considering the mechanism, energetics, and kinetics of the major PFPs. Evidence proving semi-quantitative predictability of some of the ion intensity relationships (IIRs) of the MS/MS spectra of protonated peptides is presented.     

Hypoxia-induced pathways in breast cancer

Hypoxia, a common consequence of solid tumor growth in breast cancer and other cancers, serves to propagate a cascade of molecular pathways which include angiogenesis, glycolysis, and alterations in microenvironmental pH. Hypoxia-inducible factors, heterodimeric DNA binding complexes composed of two subunits, provide critical regulation of this response. This review presents a synopsis of the genes induced by hypoxia in the context of breast cancer and discusses how upregulation of HIF-1 activity, and the homologous factor HIF-2, are not only fundamental for the adaptation to hypoxia but also may be critical for tumor progression.     

Phagocytosis of apoptotic cells by liver: a morphological study

The present review deals with the morphological features of the removal of apoptotic cells by liver. The engulfment of cells undergoing apoptosis can be considered a specialized form of phagocytosis, playing a major role in the general tissue homeostasis in physiological and pathological conditions. In fact, defects of phagocytosis of apoptotic cells might have deleterious consequences for neighboring healthy cells, i.e., pathogenesis of inflammatory disease or dysregulation of the immune system. Phagocytosis of apoptotic cells by liver is a complex phenomenon, involving multiple molecular mechanisms of recognition (i.e., lectin-like receptors and receptors for externalized phosphatydilserine) of both parenchymal (hepatocytes) and nonparenchymal (Kupffer and endothelial cells) liver cells, often operating in cooperation. The data discussed in the present review are drawn from studies of phagocytosis of apoptotic cells in the liver, carried out with in vivo and in situ adhesion experiments as well as in vitro assays. Our results indicate that the three main liver cell types (hepatocytes, Kupffer, and endothelial cells) are able to recognize and internalize apoptotic cells by means of specific receptors (galactose and mannose-specific receptor; receptor for phosphatydilserine) and by cytoskeletal reorganization that favors the engulfment of the apoptotic cells. The "flags" for the identification of apoptotic cells by the liver are modifications of the surface of dead cells, i.e., sugar residues and phosphatydilserine exposition. Vitronectin receptor is not involved in such a recognition. The adhesions between modified cell surfaces of apoptotic cells and phagocytes generate cytoplasmatic signaling pathways that drive apoptotic cells to their final fate within the phagocytes (i.e., lysosomal digestion).     

A multiple technical approach to the study of apoptotic cell micronuclei

Apoptotic micronuclei have been studied, in different cell types, from a morphologic and functional point of view. Conventional electron microscopy, in various staining conditions, selective cytochemistry for DNA, and freeze fracture for the analysis of chromatin fiber organization and size were performed. In situ TdT and Pol I immunofluorescent techniques were carried out to detect double- and single-strand DNA breaking points by confocal laser scanning microscopy. Apoptotic cell ultrathin cryosections were also performed and were analysed by field emission in lens scanning electron microscopy. Double/single strand massively cleaved DNA was detected in micronuclei, with a highly supercoiled, uniformly packed, very dense arrangement.     

Endocytic pathways in the olfactory and vomeronasal epithelia of the mouse: ultrastructure and uptake of tracers.

Mammalian olfactory neurons possess a well-developed system of endocytic vesicles, endosomes, and lysosomes in their dendrites and perikarya. Vomeronasal neurons are similar and also contain much perikaryal agranular endoplasmic reticulum (AER). Olfactory supporting cells contain endocytic vesicles and endosomes associated closely with abundant fenestrated AER, and vesicles and numerous large dense vacuoles are present basally. Vomeronasal supporting cells have little AER, and few dense vacuoles occur in their bases. In olfactory neurons, ultrastructural tracers (0.08% horseradish peroxidase, thorium dioxide, ferritin) are endocytosed by olfactory receptor endings and transported to the cell body, where their movement is halted in lysosomes. Higher concentrations (1%) of horseradish peroxidase penetrate olfactory receptor plasma membranes and intercellular junctions. In olfactory supporting cells, endocytosed tracers pass through endosomes to accumulate in dense basal vacuoles. These observations indicate that olfactory sensory membranes are rapidly cycled and that endocytosed materials are trapped within the epithelium. It is proposed that in the olfactory epithelium, endocytosis presents redundant odorants to the enzymes of the supporting cell AER to prevent their accumulation, whereas in the vomeronasal epithelium the receptor cells carry out this activity.     

Mitochondrial alterations and autofluorescent conversion of Candida albicans induced by histatins

The mechanism of the candidacidal activity of histatins 3 and 5 (Hst) is still a matter of debate. Previous studies have indicated that Hst induce cell permeabilization, generation of reactive oxygen species (ROS) by mitochondria, inhibition of the respiratory chain, and energy-dependent cytotoxic release of ATP. On the other hand, the multiplicity of effects and the apparent contrast between experimental data continue to render the mechanism of Hst-induced killing of C. albicans unclear. In this investigation, using fluorescent probes (the potential-sensitive mitochondrial probe tetramethylrhodamine methyl ester perchlorate, TMRM; the ROS-sensitive probe dihydrofluorescein diacetate, DHF; the membrane-impermeant probe, calcein) and autofluorescence data we observed that Hst induce ROS generation by mitochondria undergoing a high energy swelling condition, accompanied by oxidation of cytosolic NAD(P)H and mitochondrial flavoproteins. ROS generation and swelling, attributable to an inhibition of the respiratory chain and to impairment of the K/H-exchanger, were followed by mitochondrial depolarization. Mitochondrial changes were accompanied by massive calcein influx, indicative of cell permeabilization, and prominent alterations of the cell size, shape, and optical density. The loss of proliferative activity was correlated, on a single cell basis, to the acquisition of a lipofuscin-like autofluorescence.     

Defining mitogen-activated protein kinase pathways with mass spectrometry-based approaches

Mitogen-activated protein kinases are a group of ubiquitously expressed kinase pathways that have been conserved from yeast through humans. They control a large number of critical cell functions. Identification of targets of those kinases is necessary to define signal transduction pathways that lead to cell responses. The application of a number of mass spectrometry-based techniques to the identification of phosphoproteins is reviewed. A new proteomic approach is described for the identification of the downstream targets of specific kinases that combines phosphorylation of cell lysates in in vitro kinase reactions by active recombinant kinase with protein separation by two-dimensional (2D) gel electrophoresis or SDS-PAGE and phosphoprotein identification by MALDI-TOF mass spectrometry or by phosphopeptide enrichment and tandem mass spectrometry. The results suggested that a combination of multiple approaches will be required to fully identify phosphoproteomes.     

Ultrastructural study of apoptotic U937 [corrected] cells treated with different pro-apoptotic substances

Human monocytic leukemia U937 cells readily undergo apoptosis when exposed to various stimuli, including inhibition of protein synthesis, oxidative stress, antitumoral agents, etc. The sequential, step-by-step morphological changes in U937 cells that occur during the apoptotic program are largely determined by the activation of a specific class of proteases, the caspases. The action of these proteases were followed at the ultrastructural level. From our observations 1) no unique morphological feature exists during apoptosis, even in the same cell type; 2) the extent of the morphological modifications are inducer- and dose-dependent; 3) double or triple treatments amplify the morphological modifications with a single inducer, but not the rate of apoptosis; and 4) in the case of double treatment the second inducer has to have a cytoplasmatic target because damage to the cytoplasm occurs before nuclear modifications become visible. These data should facilitate a more objective evaluation of apoptosis in conditions where antiproliferative drugs, like antiblastic or immunosuppressive molecules, are used to monitor the efficiency of treatment.     

L-Selenocystine induce HepG2 cells apoptosis through ROS-mediated signaling pathways

At present, Hepatocarcinoma is one of the main causes of tumor related death all over the world. However, there are still many clinical restrictions on the treatment of liver cancer. Recently, L-Selenocystine has been shown to be a novel treatment for tumors, especially human glioma cells. But, the mechanism of L-Selenocystine against hepatocellular carcinoma remains unclear. Therefore, the main objective of this study was to investigate the effects of L-Selenocystine on HepG2 cell proliferation and activation of reactive oxygen species (ROS) mediated signaling pathway. L-Selenocystine can significantly inhibit HepG2 cell proliferation by activating caspase-3 and cleaving PARP to induce apoptosis. Moreover, the excessive production of ROS and the influence of Bax signaling pathway which can promote cell apoptosis are key factors for L-Selenocystine to induce HepG2 cell apoptosis. Therefore, the date of this study suggest that ROS mediated signal transduction mechanism may provide certain reference significance for L-Selenocystine induced HepG2 cell apoptosis.     

Raloxifene-loaded and aptamer-bonded exosomes induce autophagic and apoptotic death in HeLa cells by enhancing the lysosomotropic effect

Background: Raloxifene, a selective estrogen receptor modulator, is also known to be a lysosomotropic agent. The bioavailability of raloxifene is around 2% due to extensive hepatic transport. Exosomes are nanosized vesicles that are naturally released from cells. Method: In this study, exosomes released from HeLa cervical cancer cells were loaded with raloxifene to increase its bioavailability, and an aptamer was attached to the exosome membrane for targeting only HeLa cells. Characterization of exosomes isolated from HeLa cells was performed by transmission electron microscopy, zeta sizer, and western blotting. In addition, the cytotoxic, apoptotic, autophagic, and lysosomotropic effects of the prepared Exo-Apt-Ral formulation on HeLa cervical cancer cells were investigated. Results: According to zeta analysis, the sizes of the empty exosome and Exo-Apt-Ral formulation were measured as 66 ± 12 and 120 ± 21 nm, respectively. There was a rise in the lysosomal permeability of HeLa cells after the Exo-Apt-Ral application. In addition, both apoptotic and autophagic death mechanisms were triggered in HeLa cells after the Exo-Apt-Ral application. Conclusion: This study showed that raloxifene functionalized by loading into aptamer-bound exosomes can be a new targeted drug carrier system for cervical cancer.     

The complexity of the visual cells and visual pathways of the sturgeon

The visual cells in the retinae of the sturgeon were studied by scanning electron microscopy and transmission electron microscopy. Our investigations revealed the presence of rods, two types of single cones, one type of double cone (two nonidentical cone components adhered together), and one type of twin cone (two identical cone components adhered together). In some of the cones, large glycogen bodies were present in the inner segments and all cones contained oil droplets. Such cone morphology was very similar to that described in the retinae of higher vertebrates, for example the chicken. DiI tracing of retinofugal pathways following uniocular injection demonstrated their bilateral localization and extensive termination in the diencephalon and mesencephalon of both sides. Fibers also crossed over from one side to another through commissures, including the posterior commissure. The complexity of the pathway surpassed that of the teleosts and further indicated the evolutionary importance of this fish.