The significance of macroautophagy in health and disease

During the past decade there has been a substantial increase in macroautophagy (herein simply referred to as autophagy) research due to a growing understandingof this process, coupled with improved new techniques for its detection. Autophagy (auto — self, phagy — eating) is defined as a fundamental lysosomalcatabolic pathway responsible for degrading long-lived proteins, protein aggregates, oxidised lipids, damaged organelles, and even microbial invaders. Although autophagy occurs at basal levels in normal conditions, many different forms ofmetabolic stress, including starvation, hypoxia, high temperature, high culturedensity, hormones, and growth factor deprivation can dramatically stimulatean autophagic response. Autophagy plays a critical role in maintaining cellularhomeostasis and genomic integrity and therefore has been implicated in manyphysiological activities such development, differentiation, and tissue remodelling.Consequently, defects in autophagy have been linked to various human diseasessuch as neurodegenerative and muscle disorders, cancers, cardiac failure, andinflammatory disorders. This mini-review summarises current knowledge in a fieldof mammalian autophagy and considers the significance of autophagy in humanphysiology and pathology

Ultrastructural aspects of acute pancreatitis induced by 2, 2'-azobis (2-amidinopropane) dihydrochloride (AAPH) in rats

Background. Pathophysiology of acute pancreatitis (AP) has not been clearly established, nevertheless accumulating evidence implicates highly reactive oxygen species (ROS) as important mediators of exocrine tissue damage.Methods. In this study, we used a water-soluble radical initiator, 2,2'-azobis-(2-amidinopropane) dihydrochloride (AAPH), to investigate consequences of oxidative stress insult to rats pancreas. Detailed ultrastructural characterization of exocrine pancreatic changes that involved a time course of AAPH (40 mg/1kg body weight) induction from 3 to 24 hours was performed. Results. Considerable damage to the mitochondria in acinar cells manifested by increased translucence of the matrix, partial destruction of cristae and formation of myelin figures were noted. At the same time, focal dilation, degranulation of rough endoplasmic reticulum (RER), and reduced number of zymogen granules was observed. The most prominent ultrastructural feature was accumulation of highly polymorphic cytoplasmic vacuoles in acinar cells. Different in size and shape double membrane-bound autophagosomes with sequestered organelles, autophagolisosomes, and also large, empty, single-membrane-bound vacuoles were observed within the cytoplasm. Conclusions. The results indicate that intensive but impaired autophagy mediates pathological accumulation of vacuoles in acinar cells. Rat model of acute pancreatitis induced by AAPH  is useful to investigate the early events of oxidative stress insult to pancreas

Controlled cholesterol efflux from the aortic smooth muscle cells triggers microheterogeneity of plasma membrane lipids and induces modification of the mitochondrial topology

It is generally accepted that phospholipids of plasma membrane display lateral segregation into small microdomains commonly known as lipid rafts. Such lateral lipid organization is under the control of cholesterol. Cholesterol depletion evolved by methyl-β-cyclodextrin (MCD) has been found to induce further marked perturbation in lateral lipid organization, evidenced in the high field part of electron paramagnetic resonance spectra of plasma membranes labelled with a spectroscopic probe, namely 5-doxyl-stearic acid (5DOXS). Such perturbation of surface lipid topo-logy has been found to induce distinct changes in the mitochondrial morpho-logy, i.e. switch from filamentous form into small granular form

4-OH-TEMPO prevents the morphological alteration of rat thymocytes primed to apoptosis by oxidative stress inducer ButOOH

Thymocytes exposed to the pro-oxidant tert–butyl-hydroperoxide (ButOOH) display a number of dramatic changes in morphology similar to those observed in the case of dexamethasone-treated cells. Both reagents induce nuclear chromatin peripheral aggregation below the nuclear membrane. Some nuclei themselves break up producing two or more fragments. ButOOH-treated cells are morphologically characterised by cell shrinkage, extensive surface blebbing and, finally, fragmentation into membrane–bound apoptotic bodies composed of cytoplasm and tightly packed with or without nuclear fragments. An increased level of lipid hydroxyperoxides was detected after exposure of thymocytes to ButOOH. Both oxidative stress markers and morphological damage to cells were prevented by the antioxidant 4-OH-TEMPO

Are early somatic embryos of the norway spruce (Picea abies (L.) Karst.) organised?

Background Somatic embryogenesis in conifer species has great potential for the forestry industry. Hence, a number of methods have been developed for their efficient and rapid propagation through somatic embryogenesis. Although information is available regarding the previous process-mediated generation of embryogenic cells to form somatic embryos, there is a dearth of information in the literature on the detailed structure of these clusters. Methodology/Principal Findings The main aim of this study was to provide a more detailed structure of the embryogenic tissue clusters obtained through the in vitro propagation of the Norway spruce (Picea abies (L.) Karst.). We primarily focused on the growth of early somatic embryos (ESEs). The data on ESE growth suggested that there may be clear distinctions between their inner and outer regions. Therefore, we selected ESEs collected on the 56th day after sub-cultivation to dissect the homogeneity of the ESE clusters. Two colourimetric assays (acetocarmine and fluorescein diacetate/propidium iodide staining) and one metabolic assay based on the use of 2,3,5-triphenyltetrazolium chloride uncovered large differences in the metabolic activity inside the cluster. Next, we performed nuclear magnetic resonance measurements. The ESE cluster seemed to be compactly aggregated during the first four weeks of cultivation; thereafter, the difference between the 1H nuclei concentration in the inner and outer clusters was more evident. There were clear differences in the visual appearance of embryos from the outer and inner regions. Finally, a cluster was divided into six parts (three each from the inner and the outer regions of the embryo) to determine their growth and viability. The innermost embryos (centripetally towards the cluster centre) could grow after sub-cultivation but exhibited the slowest rate and required the longest time to reach the common growth rate. To confirm our hypothesis on the organisation of the ESE cluster, we investigated the effect of cluster orientation on the cultivation medium and the influence of the change of the cluster’s three-dimensional orientation on its development. Maintaining the same position when transferring ESEs into new cultivation medium seemed to be necessary because changes in the orientation significantly affected ESE growth. Conclusions and Significance This work illustrated the possible inner organisation of ESEs. The outer layer of ESEs is formed by individual somatic embryos with high metabolic activity (and with high demands for nutrients, oxygen and water), while an embryonal group is directed outside of the ESE cluster. Somatic embryos with depressed metabolic activity were localised in the inner regions, where these embryonic tissues probably have a very important transport function

Titanium dioxide nanoparticles enhance production of superoxide anion and alter the antioxidant system in human osteoblast cells

Karolina Niska,1 Katarzyna Pyszka,1 Cecylia Tukaj,2 Michal Wozniak,1 Marek Witold Radomski,3–5 Iwona Inkielewicz-Stepniak1 1Department of Medical Chemistry, 2Department of Electron Microscopy, Medical University of Gdansk, Gdansk, Poland; 3School of Pharmacy and Pharmaceutical Sciences, Trinity Biomedical Sciences Institute, The University of Dublin Trinity College, Dublin, Ireland; 4Kardio-Med Silesia, 5Silesian Medical University, Zabrze, Poland Abstract: Titanium dioxide (TiO2) nanoparticles (NPs) are manufactured worldwide for a variety of engineering and bioengineering applications. TiO2NPs are frequently used as a material for orthopedic implants. However, to the best of our knowledge, the biocompatibility of TiO2NPs and their effects on osteoblast cells, which are responsible for the growth and remodeling of the human skeleton, have not been thoroughly investigated. In the research reported here, we studied the effects of exposing hFOB 1.19 human osteoblast cells to TiO2NPs (5–15 nm) for 24 and 48 hours. Cell viability, alkaline phosphatase (ALP) activity, cellular uptake of NPs, cell morphology, superoxide anion (O2•-) generation, superoxide dismutase (SOD) activity and protein level, sirtuin 3 (SIR3) protein level, correlation between manganese (Mn) SOD and SIR, total antioxidant capacity, and malondialdehyde were measured following exposure of hFOB 1.19 cells to TiO2NPs. Exposure of hFOB 1.19 cells to TiO2NPs resulted in: (1) cellular uptake of NPs; (2) increased cytotoxicity and cell death in a time- and concentration-dependent manner; (3) ultrastructure changes; (4) decreased SOD and ALP activity; (5) decreased protein levels of SOD1, SOD2, and SIR3; (6) decreased total antioxidant capacity; (7) increased O2•- generation; and (8) enhanced lipid peroxidation (malondialdehyde level). The linear relationship between the protein level of MnSOD and SIR3 and between O2•- content and SIR3 protein level was observed. Importantly, the cytotoxic effects of TiO2NPs were attenuated by the pretreatment of hFOB 1.19 cells with SOD, indicating the significant role of O2•- in the cell damage and death observed. Thus, decreased expression of SOD leading to increased oxidizing stress may underlie the nanotoxic effects of TiO2NPs on human osteoblasts. Keywords: TiO2NPs, superoxide dismutase, sirtuin 3, nanotoxicit