Luigi Sciola

Enhancing effect of manganese on L-DOPA-induced apoptosis in PC12 cells: role of oxidative stress.

Abstract : L‐DOPA and manganese both induce oxidative stress‐mediated apoptosis in catecholaminergic PC12 cells. In this study, exposure of PC12 cells to 0.2 mM MnCl2 or 10‐20 μM L‐DOPA neither affected cell viability, determined by the 3‐(4,5‐dimethylthiazol‐2‐yl)‐2,5‐diphenyltetrazolium bromide (MTT) assay, nor induced apoptosis, tested by flow cytometry, fluorescence microscopy, and the TUNEL technique. L‐DOPA (50 μM) induced decreases in both cell viability and apoptosis. When 0.2 mM MnCl2 was associated with 10, 20, or 50 μM L‐DOPA, a concentration‐dependent decrease in cell viability was observed. Apoptotic cell death also occurred. In addition, manganese inhibited L‐DOPA effects on dopamine (DA) metabolism (i.e., increases in DA and its acidic metabolite levels in both cell lysate and incubation medium). The antioxidant N‐acetyl‐L‐cysteine significantly inhibited decreases in cell viability, apoptosis, and changes in DA metabolism induced by the manganese association with L‐DOPA. An increase in autoxidation of L‐DOPA and of newly formed DA is suggested as a mechanism of manganese action. These data show that agents that induce oxidative stress‐mediated apoptosis in catecholaminergic cells may act synergistically.

Prevention of Diabetes-Induced Microangiopathy by Human Tissue Kallikrein Gene Transfer

Background—Microvascular insufficiency represents a major cause of end-organ failure among diabetics. Methods and Results—In streptozotocin-induced diabetic mice, we evaluated the potential of human tissue kallikrein (hTK) gene as a sole therapy against peripheral microangiopathy. Local delivery of hTK gene halted the progression of microvascular rarefaction in hindlimb skeletal muscle by inhibiting apoptosis, thus ensuring an improved hemodynamic recovery in case of supervening vascular occlusion. The curative action of hTK did not necessitate insulin supplementation. Application of gene therapy at a stage of established microangiopathy stimulated vascular regeneration. Conclusions—Our studies indicate that hTK may represent a useful tool for the treatment of microvascular complications in diabetics.

Activation signals of T lymphocytes in microgravity

Human peripheral blood lymphocytes and monocytes were activated with concanavalin A with or without exogenous recombinant interleukin 1 (IL-1) alone or IL-1 + interleukin 2 (IL-2) under microgravity conditions to test the hypothesis that lack of production of IL-1 by monocytes is the cause of the near total loss of activation observed earlier on several Spacelab flights. The 60 min failure of the on-board 1 x g reference centrifuge at the time of the addition of the activator renders the in-flight data at 1 x g unreliable. However, the data from a previous experiment on SLS-1 show that there is no difference between the results from the in-flight 1 x g centrifuge and 1 x g on ground. The comparison between the data of the cultures at 0 x g in space and of the synchronous control at 1 x g on ground show that exogenous IL-1 and IL-2 do not prevent the loss of activity (measured as the mitotic index) at 0 x g; production of interferon-gamma, however, is partially restored. In contrast to a previous experiment in space, the production of IL-1 is not inhibited.

Movements and interactions of leukocytes in microgravity.

The mitogenic activation of human lymphocytes resuspended in vitro is dramatically reduced in microgravity. As cell-cell contacts are one of the elements essential for activation, the behaviour of human leukocytes (mainly lymphocytes and monocytes as accessory cells) in the presence of the mitogen concanavalin A was studied in the centrifuge microscope NIZEMI at 0 x g. Aggregates (formed by intercellular bindings of membrane glycoproteins via the tetravalent alpha-glucoside ligand concanavalin A) were found at 0 x g as well as at 1 x g already 12 h after the addition of the mitogen. In general, the aggregates observed at 0 x g after an incubation time of 46 and 78 h were smaller than the corresponding aggregates in the ground control. The findings are of primary importance since they confirm the indirect evidence we had from earlier Spacelab experiments and demonstrate that cell-cell contacts are occurring also in microgravity. In addition, single cells in 0 x g show a significant higher locomotion velocity than the cells at 1 x g. The fact that the locomotion capability is not decreased during the 78-h incubation with concanavalin A provides further evidence that the cells are not proceeding through the cell cycle.

Static Cytofluorometry and Fluorescence Morphology of Mitochondria and DNA in Proliferating Fibroblasts

The shape, distribution, and content of mitochondria in individual cells were examined during the cell cycle phases (G(0)/G(1), S, G(2) mitosis) in living human fibroblasts by static cytofluorometry and fluorescence microscopy. The morphocytochemical evaluations were performed in cell cultures submitted to double supravital fluorochrome staining with Hoechst 33342 and DiOC(6) to label DNA and mitochondria, respectively. The staining modalities were based on the stability of mitochondrial labeling. The G(1) to early S phases were characterized by the presence of filamentous mitochondria, except during the early postmitotic period. During late S, G(2), and mitotic phases, mitochondrial mass reached its highest value and mitochondria became short and numerous. During the last stage of mitosis, mitochondria were distributed among daughter cells through a cytoplasmic bridge.

Influence of microgravity on mitogen binding and cytoskeleton in Jurkat cells

The effects of microgravity on Jurkat cells--a T-lymphoid cell line--was studied on a sounding rocket flight. An automated pre-programmed instrument permitted the injection of fluorescent labelled concanavalin A (Con A), culture medium and/or fixative at given times. An in-flight 1 g centrifuge allowed the comparison of the data obtained in microgravity with a 1 g control having the same history related to launch and re-entry. After flight, the cells fixed either at the onset of microgravity or after a or 12 minute incubation time with fluorescent concanavalin A were labelled for vimentin and actin and analysed by fluorescence microscopy. Binding of Con A to Jurkat cells is not influenced by microgravity, whereas patching of the Con A receptors is significantly lower. A significant higher number of cells show changes in the structure of vimentin in microgravity. Most evident is the appearance of large bundles, significantly increased in the microgravity samples. No changes are found in the structure of actin and in the colocalisation of actin on the inner side of the cell membrane with the Con A receptors after binding of the mitogen.

Channelling of deoxyribose moiety of exogenous DNA into carbohydrate metabolism: role of deoxyriboaldolase

In bacteria, the addition of (deoxy)nucleosides or (deoxy)ribose to the growth medium causes induction of enzymes involved in their catabolism, leading to the utilisation of the pentose moiety as carbon and energy source. In this respect, deoxyriboaldolase appears the key enzyme, allowing the utilisation of deoxyribose 5-P through glycolysis. We observed that not only deoxynucleosides, but also DNA added to the growth medium of Bacillus cereus induced deoxyriboaldolase; furthermore, the switch of the culture from aerobic to anaerobic conditions caused a further increase in enzyme activity, leading to a more efficient channelling of deoxyribose 5-P into glycolysis, probably as a response to the low energy yield of the sugar fermentation. In eukaryotes, the catabolism of (deoxy)nucleosides is well known. However, the research in this field has been mainly devoted to the salvage of the bases formed by the action of nucleoside phosphorylases, whereas the metabolic fate of the sugar moiety has been largely neglected. Our results indicate that the deoxyriboaldolase activity is present in the liver of several vertebrates and in a number of cell lines. We discuss our observations looking at the nucleic acids not only as informational molecules, but also as a not negligible source of readily usable phosphorylated sugar.

PMA withdrawal in PMA‐treated monocytic THP‐1 cells and subsequent retinoic acid stimulation, modulate induction of apoptosis and appearance of dendritic cells

To analyse proliferation, differentiation and apoptosis in THP‐1 cells after stimulation with phorbol 12‐myristate 13‐acetate (PMA) and retinoic acid (RA).

Cytotoxicity of lazaroid U-75412E in human epithelial cell line (Wish)

The 21-aminosteroids (or lazaroids) are a recently synthesized class of compounds demonstrated to protect tissue against damage induced by trauma and/or ischemia. Currently, very little is known about the biological effects of lazaroids. In this work the action of lazaroid U-75412E on a human epithelial cell line (Wish) was evaluated. The data obtained showed an inhibition of cell growth and a dose- and time-dependent decrease of cell viability. Furthermore, a dose- and time-dependent increase of cells in the G2/M phase with the appearance of apoptotic cells was observed by flow cytometric analysis. Nuclear fragmentation was also evident. Lactate dehydrogenase release and scanning electron microscopy experiments suggested that plasma membrane integrity was altered by this compound. The immunofluorescence technique and transmission electron microscopy images also showed intracellular damage, such as alteration of microtubular arrangement, mitochondrial swelling and the presence of vacuoles. This study demonstrated that 1 microM U-75412E was unable to modify these parameters, while higher concentrations (6-75 microM) had a cytotoxic effect on Wish cells.

Kinetic changes of αB crystallin expression in neoplastic cells and syngeneic rat fibroblasts at various subculture stages

AbstractαB crystallin, a structural protein of the mammalian lens essential for the maintenance of lens transparency, is also expressed, at variable levels, in many extraocular tissues where it plays a protective role in stress conditions. In fact, heat or toxic shocks, as well as pathological states, increase αB crystallin levels in many cell types. Here we show that αB crystallin expression is also modulated in subcultures of rat fibroblasts and Galliera sarcoma cells. Western blots analysis with anti αB crystallin antibodies reveals the presence of the protein in both cell populations, although the kinetic pattern of expression is different. Galliera fibroblasts constitutively express the protein up to the 70th subculture and afterwards the synthesis ceases. On the other hand, Galliera sarcoma cells do not contain αB crystallin in the early stages of the culture, but there is a progressive increases between the 20th and 40th cell subculture. Differences also exist concerning the intracellular distribution: αB crystallin is diffusely localized in the cytoplasm of fibroblasts while in sarcoma cells it localizes mainly to the perinuclear region. αB crystallin is totally recovered as soluble protein in the supernatants obtained after low speed centrifugation of fibroblast homogenates, while in sarcoma cells a portion of the protein is also recovered in the insoluble pellet. Intracellular pH measurements show an alkaline cytosol in sarcoma cells compared to fibroblasts. Heat shock treatment of fibroblast subcultures constitutively expressing αB crystallin induces an over-expression of the protein, while in fibroblasts whose biosynthetic capacity is lost, heat shock is unable to activate the crystallin gene. Correlation between αB crystallin expression and proliferative rate shows that highly proliferating fibroblasts do not express αB crystallin, while neoplastic cells do.