Maria Grazia Bottone

Fluorine ion-implanted polystyrene improves growth and viability of vascular smooth muscle cells in culture.

Vascular smooth muscle cells derived from the rat aorta were cultured on unmodified or F(+) ion-implanted polystyrene (5 x 10(12) or 5 x 10(14) ions/cm(2), energy 150 keV). In 1-day-old cultures, the cells adhered to the modified polystyrene in higher numbers and over larger contact areas. Increased resistance of the cells to trypsin-mediated detachment from the growth support indicated an improved adhesion of cells to the modified polymer at later culture intervals. The cells cultured on ion-modified polymers also were larger and had a higher total protein content. By use of immunocytochemistry, several specific protein species were increased, including the cytoskeletal alpha-actin and vimentin and the plasma membrane-associated vinculin, talin, alpha-v integrins, ICAM-1, and VCAM-1, which account for stronger cell-cell and cell-extracellular matrix adhesion. The lower number of cells found floating in the medium suggests that the spontaneous detachment of cells from the modified polystyrene was lower and that the viability of the adhered cell population was higher. As was shown by the two-parameter flow-cytometric measurements of BrdU incorporation and DNA content, as well as by (3)H-thymidine autoradiography, the cell proliferation on samples modified by the dose of 5 x 10(12) ions/cm(2) was similar to that in controls; and at the dose of 5 x 10(14) ions/cm(2), it tended to be even lower. The cells grown on the polymer implanted with the dose of 5 x 10(12) ions/cm(2) responded to a new artificially created cell-free area in a confluent cell layer by more intense migration whereas at the dose of 5 x 10(14) ions/cm(2), the migration ability of cells was similar to that on the unmodified polymer. The data revealed a higher biocompatibility of ion-implanted polystyrene with vascular smooth muscle cells in culture. There was better adhesion, differentiation, and survival, and there was neither excessive migration nor proliferation.

Rearrangement of nuclear ribonucleoprotein (RNP)-containing structures during apoptosis and transcriptional arrest

Abstract The aim of this paper is to review the data in the literature concerning ribonucleoprotein components during apoptosis, where a major rearrangement of RNPs takes place. In parallel with chromatin changes, the nucleoplasmic constituents (perichromatin fibrils; perichromatin granules; interchromatin granules and nuclear bodies) as well as the nucleoli aggregate into heterogeneous clusters called HERDS, in the interchromatin space. Later, these RNP‐containing structures are extruded from the nucleus and leave the cell within cytoplasmic blebs. We propose also a role for HERDS as markers of irreversible transcriptional arrest.

Nuclear RNA Is Extruded from Apoptotic Cells

During spontaneous apoptosis of thymocytes there is extrusion of ribonucleoproteins (RNPs) from the cell. The aim of this investigation was to elucidate whether the RNP aggregates in apoptotic cells and bodies still contain RNA in an appreciable amount. We demonstrated by specific cytochemical techniques that the aggregates of nuclear RNPs extruded in the cytoplasm of spontaneously apoptotic thymocytes contain RNA in a sufficient amount to be detected cytochemically. These heterogeneous ectopic RNP-derived structures (HERDS) are formed by perichromatin fibrils, interchromatin granules, perichromatin granules, and nucleolar material. The RNA detected inside these clusters should therefore correspond to both mRNA and snRNA as well as to rRNA. We never observed DNA-contaning aggregates in the cytoplasm of apoptotic thymocytes. The presence of RNA in the HERDS that may be released from apoptotic cells suggests that the decrease in the amount of total RNA during apoptosis may be mostly linked to cellular extrusion rather than to degradation of RNA by RNase activities. Another interesting aspect of these results lies in the hypothesis of apoptosis as a possible cause for the presence of autoantibodies in the serum of patients with systemic autoimmune diseases.

DADLE induces a reversible hibernation-like state in HeLa cells.

Abstract[d-Ala(2)-d-Leu(5)-Enkephalin] (DADLE) can induce hibernation when injected into ground squirrels in summer and is able to increase the survival time of explanted organs such as liver and lung. Since cell metabolism is a target of this peptide, we have treated HeLa cells with DADLE and investigated its possible effect on transcription and proliferation as well as the resumption of metabolic activity after treatment. The labelling for Pol I, Pol II and for splicing factors such as snRNPs and SC-35 decreased after treatment as did the nucleolar labelling for UBF. In treated cells, several spherical nuclear bodies were found to be labelled for hnRNPs. In parallel, the number of proliferating cells decreased after treatment with DADLE. After recovery, there was a gradual resumption of cell function: transcription and splicing factors had a distribution similar to that of controls; proliferation resumed; nuclear bodies, representing storage sites for RNPs, disappeared.

Nuclear ribonucleoprotein-containing structures undergo severe rearrangement during spontaneous thymocyte apoptosis. A morphological study by electron microscopy.

Abstract The rearrangement of nuclear ribonucleoprotein (RNP)-containing structures during spontaneous thymocyte apoptosis has been investigated by electron microscopy. Along with chromatin margination and condensation, RNPs segregate into the central portion of the nucleus, in the form of heterogeneous clusters of granules which contain interchromatin and perichromatin granules, perichromatin fibrils, nucleolar components, and probably coiled bodies. In parallel with progressive chromatin condensation and karyorrhexis, granule clusters are then extruded into the cytoplasm and are finally released at the cell surface as membrane-bound cytoplasmic debris, sometimes in association with apparently undamaged organelles such as centrioles. It is likely that this RNP segregation may correlate with a severe impairment of protein synthesis. A similar phenomenon was observed in elongating spermatids, when transcriptional arrest is induced during the process of reversible silencing of the male genome. It may be hypothesized that segregation into heterogeneous granule clusters could be a common mechanism to remove redundant RNP-containing structures from the cell.

Resveratrol-procyanidin blend: nutraceutical and antiaging efficacy evaluated in a placebo-controlled, double-blind study

Background Skin is constantly exposed to pro-oxidant environmental stress from several sources, including air pollutants, ultraviolet solar light, and chemical oxidants. Reactive oxygen species have been implicated in age-related skin disorders. Dietary bioactive antioxidant compounds, such as polyphenols, have beneficial effects on skin health. The advantage of a nutritional administration route is that blood delivers nutraceutical bioactive compounds continuously to all skin compartments, ie, the epidermis, dermis, and subcutaneous fat. The purpose of this study was to evaluate the topical and systemic effects of a dietary supplement containing resveratrol and procyanidin on age-related alterations to the skin, the skin antioxidant pool, and systemic oxidative stress levels. Methods An instrumental study was performed in 50 subjects (25 treated with supplements and 25 with placebo) to identify clinical features induced by chronoaging or photoaging. Product efficacy was evaluated after 60 days of treatment in terms of in vivo and in situ skin hydration, elasticity, and skin roughness levels, systemic oxidative stress levels by plasmatic derivatives of reactive oxygen metabolites and oxyadsorbent tests, and extent of the skin antioxidant pool. Results After 60 days of treatment, values for systemic oxidative stress, plasmatic antioxidant capacity, and skin antioxidant power had increased significantly. Additionally, skin moisturization and elasticity had improved, while skin roughness and depth of wrinkles had diminished. Intensity of age spots had significantly decreased, as evidenced by improvement in the individual typological angle. Conclusion Nutraceutical and pharmacological intervention with a supplement characterized by a specific blend of resveratrol and procyanidin may be a promising strategy to support treatments for the reduction of skin wrinkling, as well as reducing systemic and skin oxidative stress.

Two-color fluorescence detection of Poly (ADP-Ribose) Polymerase-1 (PARP-1) cleavage and DNA strand breaks in etoposide-induced apoptotic cells

During apoptosis, the nuclear enzyme Poly(ADP-Ribose) Polymerase-1 (PARP-1) catalyzes the rapid and transient synthesis of poly(ADP-ribose) from NAD+ and becomes inactive when cleaved by caspases. The regulation of these two opposite roles of PARP-1 is still unknown. We have recently investigated PARP-1 activation/degradation in Hep-2 cells driven to apoptosis by actinomycin D. In the present work, we have extended our analysis to the effect of the DNA damaging agent etoposide, and paid attention to the relationship between PARP-1 cleavage and DNA fragmentation. An original fluorescent procedure was developed to simultaneously identify in situ the p89 proteolytic fragment of PARP-1 (by immunolabeling) and DNA degradation (by the TUNEL assay). The presence of p89 was observed both in cells with advanced signs of apoptosis (where the PARP-1 fragment is extruded from the nucleus into the cytoplasm) and in TUNEL-negative cells, with only incipient signs of chromatin condensation; this evidence indicates that PARP-1 degradation in etoposide-treated apoptotic cells may precede DNA cleavage.

Cell proliferation, apoptosis and mitochondrial damage in rat B50 neuronal cells after cisplatin treatment

Abstract.  Objectives: Cisplatin (cisPt) is used as a chemotherapeutic agent for the treatment of a variety of human tumours; more recently, it has been demonstrated that tumour cell exposure to cisPt ultimately results in apoptosis, but the mechanism by which nuclear cisPt/DNA generates the cytoplasmic cascade of events involved has not been clarified. We have investigated the effects of cisPt on proliferation in the neuronal cell line B50, with particular attention being given to understand whether mitochondria are a target of cisPt and their involvement in the apoptotic process. Materials and methods: Rat neuronal B50 cells were used to investigate the mechanisms of cisPt‐induced cytotoxicity; this line has been used as a model system for neurotoxicity in vivo. Results: Changes in proliferation, induction of apoptosis, activation of caspase‐3 and DNA fragmentation were observed in the cells, as well as morphological and biochemical alterations of mithocondria. Activation of caspase‐9 confirmed that mitochondria are a target of cisPt. Conclusion: CisPt exerts cytotoxic effects in the neuronal B50 cell line via a caspase‐dependent pathway with mitochondria being central relay stations.

The Golgi apparatus is a primary site of intracellular damage after photosensitization with Rose Bengal acetate.

The aim of the present investigation was to elucidate whether the Golgi apparatus undergoes photodamage following administration of the fluorogenic substrates Rose Bengal acetate (RBAc) and irradiation at the appropriate wavelength. Human HeLa cells were treated in culture and the changes in the organization of the Golgi apparatus were studied using fluorescence confocal microscopy and electron microscopy, after immunocytochemical labeling. To see whether the cytoskeletal components primarily involved in vesicle traffic (i.e., microtubules) might also be affected, experiments of tubulin immunolabeling were performed. After treatment with RBAc and irradiation, cells were allowed to grow in drug-free medium for different times. 24 hr after irradiation, the cisternae of the Golgi apparatus became packed, and after 48-72 hr they appeared more fragmented and scattered throughout the cytoplasm; these changes in the organization of the Golgi cisternae were confirmed at electron microscopy. Interestingly enough, apoptosis was found to occur especially 48-72 h after irradiation, and apoptotic cells exhibited a dramatic fragmentation of the Golgi membranes. The immunolabeling with anti-tubulin antibody showed that microtubules were also affected by irradiation in RBAc-treated cells.

The effect of the enkephalin DADLE on transcription does not depend on opioid receptors

Abstract[D-Ala2,D-Leu5] enkephalin (DADLE) is a synthetic peptide capable of inducing a hibernation-like state in mammals in vivo and in cultured cells in vitro. The effects of DADLE seem to be due to its binding to opioid receptors; however, it inhibits the growth of LNCaP cells, devoid of opioid receptors. We have investigated the effects of DADLE on this cell line using transmission electron microscopy, immunocytochemistry and cytometry, in order to elucidate the general mechanism(s) by which this enkephalin affects cell metabolism. We demonstrated that, similar to cell lines provided with opioid receptors, in LNCaP cells DADLE induces structural modifications of cytoplasmic and nuclear constituents, as well as a decrease in transcription and proliferation. However, DADLE does not provoke an increase in apoptotic or necrotic cell fraction, and, after removing the enkephalin from the culture medium, all effects disappear. We also demonstrated that DADLE molecules enter the cytoplasm and the nucleus of LNCaP cells, mostly binding to perichromatin fibrils and dense fibrillar component, where transcription and early splicing of pre-mRNAs and pre-rRNAs occur. In conclusion, our data demonstrate that the effect of DADLE on transcription and on cultured cells does not depend on opioid receptors. DADLE can, therefore, be envisaged as an extremely promising molecule to be used for inducing a reversible hypometabolic state in various cultured cells, without provoking cell damage or death.