1,720,986 research outputs found
Clathrin-dependent endocytosis of membrane-bound RANKL in differentiated osteoclasts
No full textBone is continuously repaired and remodelled through well-coordinated activity of osteoblasts that form new bone and osteoclasts, which resorb it. Osteoblasts synthesize and secrete two key molecules that are important for osteoclast differentiation, namely the ligand for the receptor of activator of nuclear factor κB (RANKL) and its decoy receptor osteoprotegerin (OPG). Active membrane transport is a typical feature of the resorbing osteoclast during bone resorption. Normally, one resorption cycle takes several hours as observed by monitoring actin ring formation and consequent disappearance in vitro. During these cyclic changes, the cytoskeleton undergoes remarkable dynamic rearrangement. Active cells show a continuous process of exocytosis that plays an essential role in transport of membrane components, soluble molecules and receptor-mediated ligands thus allowing them to communicate with the environment. The processes that govern intracellular transport and trafficking in mature osteoclasts are poorly known. The principal methodological problem that have made these studies difficult is a physiological culture of osteoclasts that permit observing the vesicle apparatus in conditions similar to the in vivo conditions. In the present study we have used a number of morphological approaches to characterize the composition, formation and the endocytic and biosynthetic pathways that play roles in dynamics of differentiation of mature bone resorbing cells using a tri-dimensional system of physiologic coculture
Effect of the neridronate on RANKL release in differentiated primary human osteoblasts
No full textBisphosphonates are important inhibitors of bone resorption and widely used clinically to treat osteoporosis, metabolic bone diseases and other orthopaedic disorders . Inhibiting osteoclasts via the mevalonate pathway is rec- ognized as the primary mechanism of its inhibitory action. Recent evidence suggests that bisphosphonates may regulate essential signaling molecules involved in osteoclastoge- nesis such as RANKL (receptor activator of NF- kB ligand) which are synthesized by osteoblasts. In this report we have investigated into the neridronate-osteoblast interactions in modulating essential signaling molecule such as RANKL
An increase of OPG expression in the aortas of diabetic rats may contribute to the impaired myo-relaxating activity characterizing diabetes mellitus
No full text60th Meeting of the Italian Society of Anatomy and Histology. Pavia, 15-17 Settembre 200
Biochemical and morphological changes in the nuclear matrix prepared from apoptotic HL-60 cells: effect of different stabilizing procedures.
No full textApoptotic cell death is characterized by deep morphological changes that take place in the nucleus. It is unclear whether modifications also occur in the nuclear matrix, a mainly proteinaceous structure that conceivably acts as a nuclear framework. We have investigated whether biochemical and morphological alterations of the nuclear matrix prepared from apoptotic HL-60 cells were dependent on the manipulations to which isolated nuclei were subjected before DNase I digestion and 2 M NaCl extraction. Our results showed that the stabilizing procedures employed to preserve the inner fibrogranular network and nucleolar remnants of the matrix (i.e., a 37 degrees C incubation; exposure to sodium tetrathionate at 4 degrees C; exposure to sodium tetrathionate at 37 degrees C) had no effect on the protein recovery of apoptotic nuclear matrices, which was always approximately two- to fivefold less than in control matrices. Moreover, one- and two-dimensional gel analysis of nuclear matrix proteins showed that, in apoptotic samples, striking quantitative changes were present, as compared with controls. Once again, these changes were seen irrespective of the stabilizing procedures employed. Also, transmission electron microscope analysis showed similar morphological alterations in all types of apoptotic nuclear matrices. By contrast, the immunofluorescent distribution of the 240-kDa NuMA protein seen in apoptotic samples was more sensitive to the stabilizing treatments. Our results indicate that the biochemical and morphological changes of the apoptotic nuclear matrix are largely independent of the isolation protocols and strengthen the contention that destruction of the nuclear matrix network is one of the key events leading to apoptotic nuclear destruction
The nuclear matrix and apoptosis.
No full textApoptosis is a form of active cell death, genetically encoded, that plays a key role during several physiological and pathological conditions. During the apoptotic process, striking morphological and biochemical changes take place in the cell nucleus. However, the molecular mechanisms underlying these changes have escaped clarification for many years. Recently, attention has been devoted to identifying the modifications that occur during apoptosis in the nuclear matrix, a mainly proteinaceous framework structure which is thought to play a fundamental role in organizing nuclear structure and function. In this review, we focus our attention on the biochemical and morphological changes detected in the nuclear matrix during the apoptotic process. Particular emphasis will be placed on the proteolysis that some nuclear matrix proteins undergo early during the apoptotic process, as well as on the detachment of DNA loops from the matrix by the action of endonuclease(s). Future research in this field may provide important information about the principal mechanisms that cause nuclear destruction in apoptotic cells
Early differentiating osteoclast interactions with a well suitable bone-like composite.
Full text linkOsteoclasts, as well as preosteoclasts, show different adhesion features in relationship to the substrate on which cells are grown, i.e. the formation of either podosomes belt or sealing zones. Podosomes belt forms on non bone substrates, i.e. when cells interact with glass coverslips or culture plates, whereas sealing zones form when cells grow on bone-like substrates. Podosomes belt corresponds to numerous F-actin columns arranged at the cell periphery, whereas the sealing zone could be defined as a unique large band of actin [1]. In the study of bone resorption mechanisms, the employ of bone slices is not perfectly suitable to investigate actin rearrangement due to cell-extracellular matrix (ECM) interaction, since it doesn't allow to obtain high quality preparations to be examined both by light and electronmicroscopy (TEM and SEM). In particular, TEM preparation requires demineralization which could influence the chemical properties of either bone slices or bone-like composites. Moreover, the use of bone slices as scaffold, although extensive, doesn't allow ultrastructural details that are necessary in the study of mineral resorption by monocytes or preosteoclasts [2,3]. The aim of the present study was to set up an experimental model for the study of cell-ECM interaction between either monocytes or early differentiating osteoclasts and a mineralized ECM. RAW 264.7 cells (a monocyte-macrophage cell line that can differentiate in osteoclasts) were cultured on a composite constituted by calcium phosphate and type I collagen to investigate actin polymerization and podosome formation. This bone-like composite doesn't present the mechanical bone properties, but it is constituted by the main bone components and exhibits the advantage that collagen glues the mineral phase in clusters that can be either added to cell cultures or applied on coverslips, as well as to the culture medium. Light and fluorescence microscopy, as well as TEM and SEM techniques were employed. Results showed that the use of this bone-like composite allowed to obtain useful morphological information about the resorption activity of RAW 264.7 cell line differentiating towards the osteoclastic phenotype
Localization of actin binding proteins in cells blebs
No full text61st Meeting of the Italian Society of Anatomy and Histology. Sassari, Settembre 200
Intranucleolar localization of DNA topoisomerase IIα is a distinctive feature of necrotic, but not of apoptotic, Jurkat T‐cells
No full textTwo distinct types of cell death have been described: apoptosis and necrosis. However, it is becoming increasingly clear that the differences between these two types are far less numerous than initially thought. Morphological analyses might provide important information to distinguish apoptotic from necrotic samples. We recently reported that in necrotic, but not apoptotic, HL-60 human myeloid leukaemia cells, the nuclear protein topoisomerase Hot concentrated in nucleoli. In order to ascertain whether or not this phenomenon was restricted to a peculiar cell type or could be detected also in cells of lymphoid lineage, we performed an investigation aimed at defining the localization of topoisomerase IIalpha in apoptotic and necrotic Jurkat human T lymphoblastoid cells. Immunofluorescence staining demonstrated that topoisomerase IIalpha was excluded from the condensed chromatin of apoptotic cells, whereas in necrotic cells it was localized in discrete nuclear dots. Immuno-electron microscopy analysis showed that topoisomerase IIalpha was undetectable in nucleoli of normal and apoptotic cells, whereas it was present in the nucleolus of necrotic cells irrespectively of the type of inducer used (ethanol, H2O2, HgCl2). Taken together, our findings identify topoisomerase IIalpha as a potential morphological marker useful to discriminate between apoptotic and necrotic cells. (C) 2003 Wiley-Liss, Inc
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