Liliana Bruzzone

A possible role of oxidative stress in the vanadium-induced cytotoxicity in the MC3T3E1 osteoblast and UMR106 osteosarcoma cell lines.

The cytotoxicity and free radical production induced by vanadium compounds were investigated in an osteoblast (MC3T3E1) and an osteosarcoma (UMR106) cell lines in culture. Vanadate induced cell toxicity, reactive oxygen species (ROS) formation and thiobarbituric acid reactive substances (TBARS) increased in a concentration-dependent manner (0.1-10 mM) after 4 h. The concentration-response curve of vanadate-induced cytotoxicity and oxidative stress in MC3T3E1 cells was shifted to the left of the UMR106 curve, suggesting a greater sensitivity of the non-transformed cells in comparison to the osteosarcoma UMR106 cells. Supplementing with vitamin E acetate (80 microM) significantly inhibited ROS and TBARS formation but did not improve the vanadate-dependent decrease in cell number. Other vanadium compounds (vanadyl, pervanadate, and VO/Aspi, a complex of vanadyl(IV) with aspirin) showed different degrees of cell toxicity and induced oxidative stress. Altogether these results suggest that oxidative stress is involved in vanadium induced osteoblastic cytotoxicity, although the mechanism is unknown.

Non-enzymatic glycosylation of a type I collagen matrix: effects on osteoblastic development and oxidative stress

BackgroundThe tissue accumulation of protein-bound advanced glycation endproducts (AGE) may be involved in the etiology of diabetic chronic complications, including osteopenia. The aim of this study was to investigate the effect of an AGE-modified type I collagen substratum on the adhesion, spreading, proliferation and differentiation of rat osteosarcoma UMR106 and mouse non-transformed MC3T3E1 osteoblastic cells. We also studied the role of reactive oxygen species (ROS) and nitric oxide synthase (NOS) expression on these AGE-collagen mediated effects.ResultsAGE-collagen decreased the adhesion of UMR106 cells, but had no effect on the attachment of MC3T3E1 cells. In the UMR106 cell line, AGE-collagen also inhibited cellular proliferation, spreading and alkaline phosphatase (ALP) activity. In preosteoblastic MC3T3E1 cells (24-hour culture), proliferation and spreading were significantly increased by AGE-collagen. After one week of culture (differentiated MC3T3E1 osteoblasts) AGE-collagen inhibited ALP activity, but had no effect on cell number. In mineralizing MC3T3E1 cells (3-week culture) AGE-collagen induced a decrease in the number of surviving cells and of extracellular nodules of mineralization, without modifying their ALP activity. Intracellular ROS production, measured after a 48-hour culture, was decreased by AGE-collagen in MC3T3E1 cells, but was increased by AGE-collagen in UMR106 cells. After a 24-hour culture, AGE-collagen increased the expression of endothelial and inducible NOS, in both osteoblastic cell lines.ConclusionsThese results suggest that the accumulation of AGE on bone extracellular matrix could regulate the proliferation and differentiation of osteoblastic cells. These effects appear to depend on the stage of osteoblastic development, and possibly involve the modulation of NOS expression and intracellular ROS pathways.

EFFECTS OF ADVANCED GLYCATION END-PRODUCTS ON THE PROLIFERATION AND DIFFERENTIATION OF OSTEOBLAST-LIKE CELLS

Two different lines of osteoblast-like cells were used to investigate the effect of advanced glycation end-products of bovine serum albumin on cell proliferation and differentiation. These parameters were found to be both dose- and time-dependent. Cell proliferation remained unchanged after a 24 h incubation period, it increased after intermediate periods of incubation with advanced glycation end-products, but was found to be depressed after several days incubation. Cellular alkaline phosphatase activity followed a similar pattern: an initial increase induced by advanced glycation end-products was generally followed, after relatively long incubation periods, by a slight but significant decrease in this parameter. 45Ca2+ uptake was only significantly inhibited by advanced glycation end-products after 24 h incubation. These results suggest that advanced glycation end-products directly regulate osteoblast proliferation and differentiation in a dose and time dependent manner.

Biological effects of a complex of vanadium(V) with salicylaldehyde semicarbazone in osteoblasts in culture: Mechanism of action

Vanadium compounds display important pharmacological actions in vivo and in vitro systems. Semicarbazones are versatile ligands with therapeutic effects. Herein, we report the effects of V(V)O(2)(salicylaldehydesemicarbazone) (V(V)-Salsem) on two osteoblast cell lines in culture (MC3T3-E1 and UMR106). V(V)-Salsem inhibited cell proliferation in a dose response manner. At 100muM, the complex caused an inhibition of ca. 48% and 38% for the normal and the tumoral osteoblasts, respectively (p<0.001). This inhibition could be partially reversed to 35% and 28% by NAC (N-acetylcysteine) and a mixture of vitamins E and C. Changes in cell proliferation correlated with morphological alterations and the disruption of actin cytoskeleton fibers. The complex also enhanced the level of ROS (reactive oxygen species) up to ca. 100% over basal in both cell lines. Activation of ERK signalling cascade was also observed. These events led to apoptosis (up to 44% in MC3T3-E1 and 33% in UMR106 cells). Scavengers of ROS and inhibitors of ERK cascade allowed to elucidate the mechanisms involved in the cytotoxicity. In conclusion, V(V)-Salsem displayed cytotoxic effects on osteoblasts in culture through the production of free radicals and the activation of ERK cascade. These mechanisms triggered the apoptotic events that conveyed to cell death.

Binding of several benzodiazepines to bovine serum albumin: Fluorescence study.

The interactions of lorazepam, oxazepam and bromazepam with bovine serum albumin (BSA) were studied by fluorescence spectrometry. The Stern-Volmer quenching constants and corresponding thermodynamic parameters DeltaH, DeltaG and DeltaS were calculated. The binding constants and the number of binding sites were also investigated. The distances between the donor (BSA) and the acceptors (benzodiazepines) were obtained according to fluorescence resonance energy transfer and conformational changes of BSA were observed from synchronous fluorescence spectra.

Significance of floral colour and scent in three Solanum sect. Cyphomandropsis species (Solanaceae) with different floral rewards

The role of scent and floral colour in three Solanum sect. Cyphomandropsis species with different floral rewards was studied. In the two studied species with pollen rewards, S. glaucophyllum Desf. and S. stuckertii Bitter, the principal advertisement to pollinators is the colour of the perianth and anthers, including areas that absorb UV light. In the last species, the scent emitted by the osmophores of the connective also plays a significant role in flower attraction. The floral perfume would be the only advertisement in species such as S. adelphum Morton, with perfume reward in which the cryptical anther colour does not contrast with that of the perianth. The internal face of the petals remains hidden. In this case no differences in the patterns of areas that absorb visible light and UV light were found. The main visitors to Solanum are species of Bombus and Augochloropsis. These pollinators visit S. glaucophyllum and S. stuckertii, species with pollen reward, but they do not visit flowers with perfume reward, for example S. adelphum.

Non-enzymatic glycosylation of alkaline phosphatase alters its biological

Hyperglycaemia in poorly controlled diabetic patients induces non-enzymatic glycosylation (glycation) of proteins, altering their structure and physiological bioactivity. Alkaline phosphatase (ALP) is a membrane-bound exoenzyme which faces the extracellular compartment. We have investigated the glycation of intestinal alkaline phosphatase in vitro and the consequences of such molecular modifications on certain structural and functional characteristics. The effect of glycation on alkaline phosphatase specific activity was determined after incubation of the enzyme with different sugars for various periods of time. The formation of early reversible glycation products was determined by the measurement of fructosamine levels, while the appearance of advanced glycation end products was estimated by spectrofluorometric analysis. A decrease in the specific activity of ALP was associated both with an increase in fructosamine levels and with the appearance of AGE-characteristic fluorescence. Changes in these parameters were found to depend on the incubation time, and on the concentration and glycating capability of the sugar employed. Co-incubation with aminoguanidine slowed down the appearance of protein-linked fluorescence, and additionally curbed the decrease in enzymatic specific activity. A significant correlation between the levels of ALP-fructosamine and ALP-advanced glycation end product was observed. Patterns of protein bands fractionated by SDS-PAGE were essentially identical for the nonglycated controls and the glycated samples. The electrophoretic mobility of the band of alkaline phosphatase on cellulose acetate gels increased as a function of the incubation time and the glycosylating power of the carbohydrate used. The present study provides evidence for the in vitro glycation of alkaline phosphatase, and for the consecutive alteration of its activity and structure.

Room-Temperature Phosphorescence of Impure Fluorene

Abstract Impurities in commercial fluorene were detected by thin-layer chromatography and identified by room-temperature phosphorimetry (RTP). Acenaphthene content was analyzed by the standard addition method by using normal RTP measurement procedures with T1(I) as the heavy atom.

Fluorescence Detection of Atenolol Using a Molecular Imprinted Polymer

Abstract A molecular imprinted polymer (MIP) for the recognition of atenolol has been synthesized using a non‐covalent approach. Finally, this MIP has been utilized as a recognition element in a flow‐through optosensing system with fluorescence detection, showing desirable sensitivity and selectivity characteristics.

Room temperature phosphorescence of the 1-bromonaphthalene/β-cyclodextrin inclusion complex: comparison between right-angle and front-face illumination geometry

The importance of correction for inner filter effects has been demonstrated in the case of the widely studied 1-bromonaphthalene/β-cyclodextrin inclusion complex by means of right-angle and front-face illumination geometry.