A novel fluorimetric method to evaluate red wine antioxidant activity

In the light of the increasing use of red wine in the diet, due to its antioxidant properties, the availability of a simple and fast method to assay the antioxidant activity of this beverage is needed. Here we measured wine antioxidant capacity (WAC) via "crocin bleaching test", wine polyphenol content (WPC), via Folin Ciocalteu method, and fluorimetric response (WF) of 13 Italian red wines. A fairly good correlation between WAC and WF (R = 0.82) was found, thus validating the fluorimetric method as a simple, fast and cheap method to evaluate the antioxidant activity of red wines

Ultrastructural and spectrophotometric study on the effects of putative triggers on aortic valve interstitial cells in in vitro models simulating metastatic calcification.

Metastatic calcification of cardiac valves is a common complication in patients affected by chronic renal failure. In this study, primary bovine aortic valve interstitial cells (AVICs) were subjected to pro-calcific treatments consisting in cell stimulation with (i) elevated inorganic phosphate (Pi = 3mM), in order to simulate hyperphosphatemic conditions; (ii) bacterial endotoxin lipopolysaccharide (LPS), simulating direct effects by microbial agents; and (iii) conditioned media (CM) derived from cultures of either LPS-stimulated heterogenic macrophages (commercial murine RAW264.7 cells) or LPS-stimulated fresh allogeneic monocytes/macrophages (bCM), simulating consequent inflammatory responses, alone or combined. Compared to control cultures, spectrophotometric assays revealed shared treatment-dependent higher values of both calcium amounts and alkaline phosphatase activity for cultures involving the presence of elevated Pi. Ultrastructurally, shared peculiar pro-calcific degeneration patterns were exhibited by AVICs from the same cultures irrespectively of the applied treatment. Disappearance of all cytomembranes and concurrent formation of material showing positivity to Cuprolinic Blue and co-localizing with silver precipitation were followed by the outcropping of such a material, which transformed in layers outlining the dead cells. Subsequent budding of these layers resulted in the formation of bubbling bodies and concentrically laminated calcospherulae mirroring those in actual soft tissue calcification. In conclusion, the in vitro models employed appear to be reliable tools for simulating metastatic calcification and indicate that hyperphosphatemic-like conditions could trigger valve calcification per se, with LPS and allogeneic macrophage-derived secretory products acting as possible calcific enhancers via inflammatory responses

Survival-related autophagic activity versus procalcific death in cultured aortic valve interstitial cells treated with critical normophosphatemic-like phosphate concentrations

Valve dystrophic calcification is a common disorder affecting normophosphatemic subjects. Here, cultured aortic valve interstitial cells (AVICs) were treated 3 to 28 days with phosphate (Pi) concentrations spanning the normal range in humans (0.8, 1.3, and 2.0 mM) alone or supplemented with proinflammatory stimuli to assess possible priming of dystrophic-like calcification. Compared with controls, spectrophotometric analyses revealed marked increases in calcium amounts and alkaline phosphatase activity for 2.0-mM-Pi-containing cultures, with enhancing by proinflammatory mediators. Ultrastructurally, AVICs treated with low/middle Pi concentrations showed an enormous endoplasmic reticulum (ER) enclosing organelle debris, so apparently executing a survival-related atypical macroautophagocytosis, consistently with ultracytochemical demonstration of ER-associated acid phosphatase activity and decreases in autophagosomes and immunodetectable MAP1LC3. In contrast, AVICs cultured at 2.0-mM Pi underwent mineralization due to intracellular release and peripheral layering of phospholipid-rich material acting as hydroxyapatite nucleator, as revealed by Cuprolinic Blue and von Kossa ultracytochemical reactions. Lack of immunoblotted caspase-3 cleaved form indicated apoptosis absence for all cultures. In conclusion, fates of cultured AVICs were crucially driven by Pi concentration, suggesting that serum Pi levels just below the upper limit of normophosphatemia in humans may represent a critical watershed between macroautophagy-associated cell restoring and procalcific cell death

LIPOXYGENASE ACTIVITY AND HYDROPEROXIDE FORMATION IN COFFEE (Coffea arabica L.) CHERRIES CULTIVATED BY DIFFERENT AGRONOMIC TECHNIQUES

It is widely accepted that biotic stress induces acidic lipoxygenase (LOX) activity mainly due to the effects of methyl jasmonate [1]. The activation of LOX pathway leads to the production of a large amount of oxidation products, which may affect the coffee organoleptic profile [2]. Hitherto the presence of LOX in coffee (Coffea arabica L.) plants is still scarcely examined [3], therefore the aim of this study was to investigate the possible involvement of this enzyme in oxidative stress of cherries from plants cultivated in organic and conventional manners. Primary and secondary oxidation products were evaluated in three separate cherry fractions: pulp, parchment seed and green coffee. Hydroperoxides, measured by HPLC, were highest in parchment seed and lowest in green coffee, but no significant difference was found between the samples obtained from the two cultivations. However, the pulp fraction from organic cultivated plants had a larger amount of primary peroxidation products, when compared to that from conventionally cultivated plants. The presence of LOX was then examined in all cherry fractions by immunochemical analysis. Only the pulp fraction showed an 84 kDa protein that exhibited a cross-reactivity against a pea LOX antibody. Subsequently, the presence of LOX was confirmed in heavy (28000 g) and light (100000 g) membrane pulp fractions by an enzymatic assay, immunochemical method and hydroperoxide formation. In both organic and conventional cultivation systems, the light membrane showed the highest LOX activity, measured as conjugated diene formation, at pH 8.0. Instead, the heavy membrane fraction highlighted a different pH optimum, which was found to be 5.5 in pulps from organic cultivations and 8.0 in the conventional ones, respectively. All the activities reported were inhibited by nordihydroguaiaretic acid (NDGA) and caffeic acid, well known LOX inhibitors. The NDGA-sensitive hydroperoxide production was simultaneously confirmed by RP-HPLC analysis. Oxidative stress was also detected by evaluating the antioxidant activity in all fractions. A lipophilic extraction, performed using dioxane:water (95:5, v/v), revealed that parchment seeds possessed the lowest antioxidant capacity. On the other hand, a significant difference in the antioxidative capacity was detected in parchment seeds from organic and conventionally cultivated plants. Taken together, these results show, for the first time, the presence of LOX in coffee cherries, whose activity was associated to membrane fractions. Furthermore, the cultivation techniques seem to induce the activation of different isoenzymes. In particular, the organic ones were subjected to an oxidative stress in coffee fruits leading to the expression of an acidic LOX activity, detectable in the pulp fraction. [1] Buzi A., et al. (2004). J. Phytopathol., 152: 34-42. [2] Kohlmann M., et al. (1999) Eur. J. Biochem. 260:885-895. [3] Rojas M.L., et al. (1993). Physiol. Mol. Plant Path., 43: 209-219

Ultrastructural characterization of calcification onset and progression in subdermally implanted aortic valves. Histochemical and spectrometric data

Detailed characterization of the subdermal model is a significant tool for better understanding of calcification mechanisms occurring in heart valves. In previous ultrastructural investigation on six-week-implantated aortic valve leaflets, modified pre-embedding glutaraldehyde-cuprolinic-blue reactions (GA-CB) enabled sample decalcification with concurrent retention/staining of lipid-containing polyanionic material, which lined cells and cell-derived matrix-vesicle-like bodies (phthalocyanin-positive layers: PPLs) co-localizing with the earliest apatite nucleation sites. Additional post-embedding silver staining (GA-CB-S) revealed PPLs to contain calcium-binding sites. This investigation concerns valve leaflets subjected to shorter implantation times to shed light on the modifications associated with PPLs generation and calcification onset/progression. Spectrometric estimations revealed time-dependent calcium increase, for unreacted samples, and copper modifications indicating an increase in acidic, non-glycanic material, for GA-CB-reacted samples. Two-day-implant thin sections showed emission and subsequent reabsorption of lamellipodium-like protrusions by cells, originating ECM-containing vacuoles, and/or degeneration stages characterized by the appearance of GA-CB-S-reactive, organule-derived dense bodies and progressive dissolution of all cell membranes. In one-week-implants, the first PPL-lined cells were found to co-exist with cells where GA-CB-S-reactive material accumulated, or exudated towards their edges, or outcropped at the ECM milieu, so acquiring PPL features. PPL-derived material was observed increasingly to affect the ECM on thin sections of one-week- to six-week-implants. These results show an endogenous source for PPLs and reveal that a peculiar cascade of cell degenerative steps is associated with valve mineralization in the subdermal model, providing new useful parameters for more reliable comparison of this experimental calcification process versus the physiological and pathological processes

Phloem cytochemical modification and gene expression following the recovery of apple plants from apple proliferation

Recovery of apple trees from apple proliferation was studied by combining ultrastructural, cytochemical, and gene expression analyses to possibly reveal changes linked to recovery-associated resistance. When compared with either healthy or visibly diseased plants, recovered apple trees showed abnormal callose and phloem-protein accumulation in their leaf phloem. Although cytochemical localization detected Ca2+ ions in the phloem of all the three plant groups, Ca2+ concentration was remarkably higher in the phloem cytosol of recovered trees. The expression patterns of five genes encoding callose synthase and of four genes encoding phloem proteins were analyzed by quantitative real-time reverse transcription- polymerase chain reaction. In comparison to both healthy and diseased plants, four of the above nine genes were remarkably upregulated in recovered trees. As in infected apple trees, phytoplasma disappear from the crown during winter, but persist in the roots, and it is suggested that callose synthesis/deposition and phloem-protein plugging of the sieve tubes would form physical barriers preventing the recolonization of the crown during the following spring. Since callose deposition and phloem-protein aggregation are both Ca2+-dependent processes, the present results suggest that an inward flux of Ca2+ across the phloem plasma membrane could act as a signal for activating defense reactions leading to recovery in phytoplasma-infected apple trees.L'articolo é disponibile sul sito dell'editore: http://www.apsjournals.apsnet.or

Critical effects of inorganic phosphate at threshold concentrations on cultured aortic valve interstitial cells. Macroautophagocytosis versus procalcific cell degeneration

The conventional threshold values ascribed to inorganic phosphate concentration ([Pi]) in diagnosing normophosphatemia range between 0.8mM and 1.45mM to 2.0mM [Pi]. In cultures mimicking metastatic calcification ([Pi]=3.0mM) a major role was found to be played by [Pi] (Pi-cultures) in priming a procalcific cell degeneration of bovine aortic valve interstitial cells (bAVICs), with mineralization enhancing subsequent to superstimulation with bacterial lipopolysaccharide (LPS) plus conditioned medium from cultured LPS-stimulated macrophages (Pi-LPS-CM-cultures) [1]. Here, bAVIC primary cultures were carried out which contained different [Pi] (0.4, 0.6, and 1.3mM in added solutions, i.e. 0.8, 1.3, and 2.0mM in final cultures), so including border concentrations on respect to hypophosphatemic- and hyperphosphatemic-like conditions. At 0.8mM and 1.3 [Pi] and for each incubation time (3, 9, 15, 21, and 28 days), bAVICs from Pi-cultures and Pi-LPS-CMcultures shared common ultrastructural features showing prominent macroautophagocytosis to occur, consistently with the immunohistochemical detection of the specific marker of mature autophagosomes MAP1-LC3A. Neither cell death signs nor appearance of calcific nodules were observed. At 2.0 [Pi], most bAVICs were affected by degenerative fragmentation as described for severe metastatic-like calcifcation, i.e. the appearence of phthalocianin- positive material outcropping at cell surface, acting as hydroxyapatite nucleator and being source of real calcospherulae. Quantitative spectrophotometric estimation of calcium amounts and alkaline phosphatase activity were consistent with the ultrastructural data, with (i) similar values for Pi-LPS-CM-cultures versus Pi-cultures and control cultures, at 0.8 and 1.3mM [Pi], and (ii) significantly higher values for Pi-LPS-CM-cultures versus Picultures and these latter versus controls, at 2.0mM [Pi]. Restriction of immunopositivity to caspase-8 to very few cells and complete immunonegativity to annexin-V suggested apoptosis to be a negligible epiphenomenon. In conclusion, the propensity of bAVICs to undergo procalcific degeneration resulted to correlate with [Pi] in such a manner that a differential discrimination of this parameter within the conventional normophosphatemic range is suggested for a proper evaluation of the risk for dystrophic valve calcification. Moreover, bacterial-derived inflammation seems to be regarded as an effective trigger for the higher normophosphatemic [Pi]

Pro-calcific responses by aortic valve interstitial cells in a novel in vitro model simulating dystrophic calcification

Etiopathogenetic mechanisms in calcific aortic valve stenosis are still poorly understood despite this being the third major cause of heart disease in western world. In prior in vitro cultures simulating metastatic calcification, pro­calcific effects on aortic valve interstitial cells (AVICs) resulted by adding bacterial endotoxin lipopolysaccharide (LPS) at high inorganic phosphate (Pi) levels. Here we accomplished improved in vitro models simulating either metastatic (Pi = 2.6 mM) or dystrophic calcification (Pi = 1.3 mM), in which LPS­stimulated bovine AVICs underwent extra-stimulation with macrophage-cytokine-containing media derived from paral­ lel cultures of allogeneic monocyte/macrophages in turn stimulated with LPS. In dystrophic calcification-like cultures, lower calcium amount was spectrometrically assessed with parallel reduced alkaline phosphatase activity with respect to metastatic calcification­like cultures, with an about three­fold slower progression of mineralization. Hydroxyapatite crystal precipitation was ultrastructurally found to correlate with AVIC degeneration processes culminating with the formation of phthalocyanin-positive lipidic layers (PPLs) at the surface of cells and cell-derived matrix-vesicle-like bodies, acting as calcium nucleators according to a pattern mirroring those we had previously found in in vivo conditions. In conclusion, an in vitro model has been devel­ oped enabling reliable simulations of the effects exerted on AVICs by putatively pro- or anti-calcific agents

In vitro reproduction and ultrastructural detection of the genesis of calcifying cell-derived structures identical to actual hydroxyapatite nucleators in calcific aortic valve stenosis

Unlike fairly good characterization gained for the major hydroxyapatite nucleators involved in physiological mineralization, i.e. matrix vesicles and apoptotic bodies, no details are available about source and genesis of so called “thick walled cell-derived-products” (CDPs) and “concentrically laminated calcospherulae” (CLCs), which are primary calcium deposition foci in ectopic calcification, including calcific aortic valve stenosis. Here, generation of these structures was successfully reproduced in vitro using an original model simulating either metastatic or dystrophic calcification, subsequent to differential inorganic phosphate supplementations to primary cultures of interstitial cells from bovine aortic valve cusps (AVICs). Superimposed bacterial infectious effects were simulated by additional stimulation with bacterial endotoxin lipopolysaccharide (LPS) and superstimulation with conditioned medium derived from cultures of bovine LPS-stimulated native monocytes/macrophages. At reverted microscope monitoring, AVICs were observed to undergo fragmentation giving rise to irregular debris and/or sporulation-like processes resulting in the formation of barely appreciable punctate bodies, concurrently with specific increase in spectrophotometrically assayed calcium amounts and alkaline phosphatase activity. Ultrastructurally, these cell-derived products showed features comparable to those characterizing CDPs and CLCs in ex vivo samples. The thick wall of CDPs was found to depend on a peculiar AVIC degenerative process culminating with outcropping of multilaminated lipid-containing phthalocyanin-positive layers (PPLs), as revealed by histochemical reactions with Cuprolinic blue. PPLs represented major calcium nucleators, as revealed by co-precipitation of hydroxyapatite needle-like crystals and co-localization of metallic silver deposition after von Kossa staining applied to electron microscopy. In addition, multilaminated PPLs of both degenerating AVICs and CDPs underwent sporulation-like budding and pinching off, so generating a lot of spherical CLC-like bodies, with many being superimposed by near radially oriented hydroxyapatite crystals. In conclusion, the way by which CDPs and CLCs form was ascertained. In addition, in-vitro reproduction of pro-calcific structures identical to those characterizing actual samples from calcified aortic valve cusps suggests the developed in vitro models to be a reliable tool for testing pro-calcific effects exterted by putative etiopathologic agents as well as anti-calcific by therapeutic compounds