Identification and Partial Characterization of Two Populations of Prostasomes by a Combination of Dynamic Light Scattering and Proteomic Analysis.

Prostasomes are vesicles secreted by prostate epithelial cells and are found in abundance in the semen. Here we characterized two different prostasome populations isolated from human seminal fluid. Prostasomes were isolated using differential centrifugation, while dynamic light scattering (DLS) was used to characterize their size and size distribution. Their protein content was analyzed using two-dimensional electrophoresis and mass spectrometry. DLS showed two distinct prostasome subpopulations in centrifuged seminal plasma, with an average hydrodynamic radius of 80 and 300 nm. The larger population was isolated after centrifugation at 20,000 × g (P20), while the smaller one was recovered at 100,000 × g (P100). The two fractions had a similar lipid composition, showing an elevated content of sphingomyelin and cholesterol. The P100 vesicles showed a significant over-expression of proteins involved in the endosomal sorting complexes required for transport (ESCRT) machinery such as Alix, TSG101, and syntenin-1. Some proteins possibly involved in prostate cancer were present only in one specific population (TMPRSS2 in P100 and VCP in P20). The different size and protein profile in the two subpopulations of prostasomes might support differential roles of the semen vesicles toward the target cells, and/or different secretion pathways from the organ of origin.

Nitric oxide depletion alters hematopoietic stem cell commitment toward immunogenic dendritic cells.

BACKGROUND: NO* is a key molecule involved in the regulation of cell survival, proliferation and differentiation in many cell types. In this study we investigated the contribution of NO* during the differentiation of human peripheral blood hemopoietic stem cells (CD34+HSCs) toward immunogenic dendritic cells (i-DCs). METHODS: We depleted autocrine NO* production, using NG-monomethyl-L-arginine monoacetate (L-NMMA) and paracrine NO', using oxy-hemoglobin (HbO2) as a NO* scavenger during in vitro differentiation of CD34+HSCs to i-DCs. We monitored the NO* level, cell proliferation, phenotype and differentiation potential. RESULTS: We found that the depletion of paracrine or autocrine NO* correlated with (I) an active proliferation state at the end of differentiation, when control cells were not proliferating; (II) a significant reduction in the expression levels of differentiative markers (CD1a and HLA-DR) with a parallel high expression of the CD34 marker (III) with a retrieved clonogenic ability compared to control cells. CONCLUSIONS: On the whole, our data indicate that the depletion of NO* during the commitment stage blocks CD34+HSC differentiation into i-DCs and maintains an undifferentiated, highly proliferating cell population, indicating/revealing a novel role for NO* in the commitment of CD34+HSCs into i-DCs. GENERAL SIGNIFICANCE: The essential finding of the present study is that NO*, produced in HSCs by NOS enzymes, may act as autocrine and paracrine effectors regulating the in vitro differentiation process of CD34+-HSCs toward i-DCs.

Production of nitric oxide by human salivary peroxidase and by bovine lactoperoxidase.

Peroxidases catalyze the oxidation of nitrite to nitrate in the presence of hydrogen peroxide. Two pathways may occur: one entailing the intermediate formation of NO(2) and the other implying the generation of peroxynitrite. The products of nitrite (NO(2) (-) ) oxidation by salivary peroxidase (SPO) and commercial bovine lactoperoxidase (LPO) are studied by utilizing an electrochemical assay that allows the direct, continuous monitoring of NO and/or NO(2) and by HPLC to assess nitrates at the end of the reaction. Dialyzed saliva and LPO, in the presence of H(2) O(2) , convert nitrite into nitrate and form some NO, with a molar ratio of 10(3) . In our experimental conditions, no NO(2) was detectable among the products of nitrite oxidation. SCN(-) inhibits NO formation and so does I(-) , although at higher concentrations. No effects are observed with Cl(-) or Br(-) . We conclude that SPO and LPO transform NO(2) (-) into nitrate-forming small amounts of NO in the presence of H(2) O(2) as an intermediate or a by-product, synthesized through the peroxynitrite pathway.

In Vitro Oxidative Stress Threatening Maize Pollen Germination and Cytosolic Ca2+ Can Be Mitigated by Extracts of Emmer Wheatgrass Biofortified with Selenium.

In this work, we studied the effects of in vitro oxidative stress applied by H2O2 to maize pollen germination and cytosolic Ca2+, taken as an experimental model to test the biological activity of extracts of emmer (Triticum turgidum L. spp. dicoccum (Schrank ex Shubler) Thell.) wheatgrass obtained from grains sprouted with distilled water, or salinity (50 mM) or selenium (45 mg L-1 of Na2SeO3). Wheatgrass extracts were obtained in two ways: by direct extraction in methanol, which represented the free phenolic fraction of extracts (Ef), and by residual content after alkaline digestion, which made it possible to obtain extracts with the bound fraction (Eb). Comparative tests on maize pollen were carried out by differently combining H2O2 and either wheatgrass extracts or pure phenolic acids (4-HO benzoic, caffeic, p-coumaric and salicylic). The cytosolic Ca2+ of maize pollen was influenced by either H2O2 or pure phenolic acids or Ef, but not by Eb. The negative effect of H2O2 on maize pollen germination and cytosolic Ca2+ was mitigated by Ef and, slightly, by Eb. The extent of the biological response of Ef depended on the sprouting conditions (i.e., distilled water, salinity or selenium). The extracts of Se-biofortified wheatgrass were the most effective in counteracting the oxidative stress.

Selective Inhibition of Wild Sunflower Reproduction with Mugwort Aqueous Extract, Tested on Cytosolic Ca2+ and Germination of the Pollen Grains.

Wild sunflower (Helianthus annuus L.) is an invasive species widely distributed in several regions of the world, where it shares a large area with domesticated sunflower. The imidazolinone-tolerant sunflower enables the control of problematic weeds (such as Xanthium spp., Brassica spp., wild sunflower) with imidazolinone herbicides (Clearfield® production system) in cultivated sunflower crops, but could facilitate the gene transfer of herbicide resistance, from cultivated sunflower to wild sunflower, generating hard-to-control weed biotypes or herbicide-resistant populations. The development of new practices that involve the selective inhibition of reproduction structures, such as pollen granules, could be an innovative strategy to minimize outcrossing and the origin of weed-crop hybrids in Clearfield® production systems. In this study, the effects of mugwort (Artemisia vulgaris L.) aqueous extract on cytosolic Ca2+ and the germination of pollen grains collected from conventional, wild and IMI-tolerant sunflower were tested. The results showed that mugwort deregulated Ca2+ homeostasis and markedly reduced the germination of conventional and wild sunflower pollen, but not IMI-tolerant pollen. The HPLC analysis revealed the presence of phenolic acids belonging to the hydroxycinnamic and benzoic classes in the mugwort extract. Hydroxycinnamic acids (caffeic and ferulic) deregulated the cytosolic Ca2+ of conventional and wild sunflower pollen, but not those which were IMI-tolerant, similar to mugwort extract. Selective inhibition of wild sunflower pollen in the Clearfield® sunflower crop contributes to a possible new weed management strategy, reducing the wild sunflower reproduction by seed, minimizing the potential risks of outcrossing with the formation of weed-crop hybrids. The Ca2+ selective chelating activity of caffeic or ferulic acids provides elements to be investigated for their possible use as an alternative to mugwort extract.

Persistence of the Effects of Se-Fertilization in Olive Trees over Time, Monitored with the Cytosolic Ca2+ and with the Germination of Pollen.

Selenium (Se) is an important micronutrient for living organisms, since it is involved in several physiological and metabolic processes. Biofortification with Se increases the nutritional and qualitative values of foods in Se-deficient regions and increases tolerance to oxidative stress in olive trees. Many studies have shown that Se, in addition to improving the qualitative and nutritional properties of EVO oil, also improves the plant's response to abiotic stress. This study addressed this issue by monitoring the effects of Se on cytosolic Ca2+ and on the germination of olive pollen grains in oxidative stress. The olive trees subjected to treatment with Na-selenate in the field produced pollen with a Se content 6-8 times higher than the controls, even after 20 months from the treatment. Moreover, part of the micronutrient was organic in selenium methionine. The higher selenium content did not produce toxic effects in the pollen, rather it antagonized the undesirable effects of oxidative stress in the parameters under study. The persistence of the beneficial effects of selenium observed over time in pollens, in addition to bringing out an undisputed adaptability of olive trees to the micronutrient, suggested the opportunity to reduce the number of treatments in the field.

The First Evidence of the Beneficial Effects of Se-Supplementation on In Vitro Cultivated Olive Tree Explants.

Selenium is an essential micronutrient that provides important benefits to plants and humans. At proper concentrations, selenium increases plant growth, pollen vitality, the shelf life of fresh products, and seems to improve stress resistance; these effects can certainly be attributed to its direct and indirect antioxidant capacity. For these reasons, in the present work, the effects of selenium at different dosages on in vitro cultivated olive explants were investigated to observe possible positive effects (in terms of growth and vigor) on the proliferation phase. The work was carried out on four different olive cultivars: "San Felice", "Canino", "Frantoio", and "Moraiolo". The explants were cultured in aseptic conditions on olive medium (OM), with the addition of 4 mg·L-1 of zeatin, 30 g·L-1 of sucrose, and 7 g·L-1 of agar. The experimental scheme included a comparison between explants grown with five different concentrations of Na2SeO4 (0, 10, 20, 40, and 80 mg L-1) added to the medium during three successive subcultures. Interesting information has emerged from the results and all varieties responded to different concentrations of Selenium. The optimal Se dosages varied for each cultivar, but in general, Se concentration between 10 and 40 mg L-1 increased fresh and dry weight of the explants and shoot lengths. Se treatment induced in all cultivars and for all dosages used an increase in total Se content in proliferated explants. Furthermore, as the subcultures proceeded, the ability of the explants to absorb Se did not diminish. The Se content ranged from 8.55 to 114.21 µg kg-1 plant DW in 'Frantoio', from 9.83 to 94.85 µg kg-1 plant DW in 'Moraiolo', from 19.84 to 114.21 µg kg-1 plant DW in 'Canino', and from 20.97 to 95.54 µg kg-1 plant DW in 'San Felice'. In general, the effect of selenium tends to decrease with the progress of subcultures and this suggests a sort of "adaptation" effect of the explants to its presence. The present study highlights for the first time the possibility of using in vitro cultures as biotechnological support to study supplementation with selenium and its effects on in vitro olive plant growth.

Effects of Megafol on the Olive Cultivar 'Arbequina' Grown Under Severe Saline Stress in Terms of Physiological Traits, Oxidative Stress, Antioxidant Defenses, and Cytosolic Ca2.

Salinity is one of the most impacting abiotic stresses regarding crop productivity and quality. Among the strategies that are attracting attention in the protection of crops from abiotic stresses, there is the use of plant biostimulants. In this study, Megafol (Meg), a commercial plant biostimulant, was tested on olive plants subjected to severe saline stress. Plants treated with salt alone showed substantial reductions in biomass production, leaf net photosynthesis (Pn), leaf transpiration rate (E), stomatal conductance (gs), and relative water content (RWC). In addition, samples stressed with NaCl showed a higher sodium (Na+) content in the leaves, while those stressed with NaCl and biostimulated with Meg increased the potassium (K+) content in the leaves, thus showing a higher K+/Na+ ratio. Salinity caused the accumulation of significant quantities of hydrogen peroxide (H2O2) and malondialdehyde (MDA) due to decreases in the activity of antioxidant enzymes, namely superoxide dismutase (SOD - EC 1.15.1.1), ascorbate peroxidase (APX - EC 1.11.1.11), guaiacol peroxidase (GPX - EC 1.11.1.9), and catalase (CAT - EC 1.11.1.6). When olive plants under saline stress were biostimulated with Meg, the plants recovered and showed physiological and biochemical traits much improved than salt stressed samples. Finally, Meg exhibited Ca2+-chelating activity in olive pollen grains, which allowed the biostimulant to exert this beneficial effect also by antagonizing the undesirable effects of hydrogen peroxide on Ca2+ metabolism.

Blood lipids in Antarctic and in temperate-water fish species.

Antarctic fish live in very cold water and have adapted to this exceptional environment. Hemoglobin is absent or very low; yet these fish still have erythrocytes, and from these we prepared ghost-like membranes. We studied for the first time the lipid composition of ghost membranes and of plasma in Antarctic fish (C. hamatus and T. bernacchii) and compared our results with those obtained for temperate-water fish (C. auratus and A. anguilla taken from Lake Trasimeno, Perugia, Italy). The membranes of Antarctic fish were richer in glycerophospholipid (especially phosphatidylethanolamine), whereas the membranes of temperate-water fish were richer in sphingomyelin. Unsaturated fatty acids were particularly abundant in Antarctic fish: C. hamatus had long-chain unsaturated fatty acid (especially C22:6 omega-3), whereas T. bernacchii had shorter unsaturated fatty acyl chains (c16:1, omega-7). On the other hand, C. auratus and A. anguilla were particularly rich in C16:0, which constituted more than one-half of the total fatty acid. Plasma lipids (both phospholipid and cholesterol) were much more abundant in temperate-water fish. The differences in phospholipid content were mainly due to choline glycerolipids. Measures of membrane fluidity inferred from the fluorescence anisotropy of DPH indicated that the membranes from Antarctic fish were more fluid at any measured temperature than those obtained from fish living in temperate waters. The ability to live in a very cold environment has therefore been achieved by the two Antarctic species tested in this paper by different strategies, but with the same results on fluidity.

Selenium-Enriched Pollen Grains of Olea europaea L.: Ca2+ Signaling and Germination Under Oxidative Stress.

Selenium (Se) shows antioxidant properties that can be exploited in plants to combat abiotic stresses caused by reactive oxygen species produced in excess (ROS). Here, we show that the Se-fertilization of olive trees with sodium selenate effectively protects the pollen from oxidative stress. Pollen isolated from plants treated with Se or from untreated controls was incubated in vitro with H2O2 to produce an oxidative challenge. Given the impact of ROS on Ca2+ homeostasis and Ca2+-dependent signaling, cytosolic Ca2+ was measured to monitor cellular perturbations. We found that H2O2 interrupted Ca2+ homeostasis only in untreated pollen, while in samples treated in vitro with sodium selenate or selenium methionine, Ca2+ homeostasis was preserved. Furthermore, germination rates were considerably better maintained in Se-fertilized pollen compared to non-fertilized pollen (30% vs. 15%, respectively) after exposure to 1 mM H2O2. The same was observed with pollen treated in vitro with Se-methionine, which is the organic form of Se, in which part of the fertigated sodium selenate is converted in the plant. Combined, our results show a close correlation between ROS, Ca2+ homeostasis, and pollen fertility and provide clear evidence that Se-fertilization is a potential approach to preserve or improve agricultural productivity.

Selenium maintains cytosolic Ca2+ homeostasis and preserves germination rates of maize pollen under H2O2-induced oxidative stress.

Selenium (Se) displays antioxidant properties that can be exploited, in plants, to counteract abiotic stresses caused by overly-produced reactive oxygen species (ROS). Here, we show that fertigation of maize crops with sodium selenate effectively protects pollen against oxidative stress. Pollen isolated from Se-treated plants (Se1) and untreated controls (Se0) was incubated in vitro with H2O2 to produce oxidative challenge. Given the impact of ROS on Ca2+ homeostasis and Ca2+-dependent signaling, cytosolic Ca2+ was measured to monitor cellular perturbations. We found that H2O2 disrupted Ca2+ homeostasis in Se0 pollen only, while Se1 samples were preserved. The same trend was observed when Se0 samples were treated with sodium selenate or Se-methionine, which recapitulated in vitro the protective capacity of Se-fertigation. Furthermore, we found that germination rates were much better retained in Se1 as compared to Se0 (46% vs 8%, respectively) after exposure to 20 mM H2O2. The same was observed with Se0 pollen treated with Se-methionine, which is the organic form of Se into which most fertigated sodium selenate converts in the plant. These results, together, show a close correlation between ROS, Ca2+ homeostasis and pollen fertility, and provide strong evidence that Se-fertigation is an excellent approach to preserve or enhance agricultural productivity.

A Na+ /Ca2+ exchanger of the olive pathogen Pseudomonas savastanoi pv. savastanoi is critical for its virulence.

In a number of compatible plant-bacterium interactions, a rise in apoplastic Ca2+ levels is observed, suggesting that Ca2+ represents an important environmental clue, as reported for bacteria infecting mammalians. We demonstrate that Ca2+ entry in Pseudomonas savastanoi pv. savastanoi (Psav) strain DAPP-PG 722 is mediated by a Na+ /Ca2+ exchanger critical for virulence. Using the fluorescent Ca2+ probe Fura 2-AM, we demonstrate that Ca2+ enters Psav cells foremost when they experience low levels of energy, a situation mimicking the apoplastic fluid. In fact, Ca2+ entry was suppressed in the presence of high concentrations of glucose, fructose, sucrose or adenosine triphosphate (ATP). Since Ca2+ entry was inhibited by nifedipine and LiCl, we conclude that the channel for Ca2+ entry is a Na+ /Ca2+ exchanger. In silico analysis of the Psav DAPP-PG 722 genome revealed the presence of a single gene coding for a Na+ /Ca2+ exchanger (cneA), which is a widely conserved and ancestral gene within the P. syringae complex based on gene phylogeny. Mutation of cneA compromised not only Ca2+ entry, but also compromised the Hypersensitive response (HR) in tobacco leaves and blocked the ability to induce knots in olive stems. The expression of both pathogenicity (hrpL, hrpA and iaaM) and virulence (ptz) genes was reduced in this Psav-cneA mutant. Complementation of the Psav-cneA mutation restored both Ca2+ entry and pathogenicity in olive plants, but failed to restore the HR in tobacco leaves. In conclusion, Ca2+ entry acts as a 'host signal' that allows and promotes Psav pathogenicity on olive plants.

Behavior of Four Olive Cultivars During Salt Stress.

Olive is considered as a moderately salt tolerant plant, however, tolerance to salt appears to be cultivar-dependent and genotypic responses have not been extensively investigated. In this work, saline stress was induced in four olive cultivars: Arbequina, Koroneiki, Royal de Cazorla and Fadak 86. The plants were grown in 2.5 l pots containing 60% peat and 40% of pumice mixture for 240 days and were irrigated three times a week with half-strength Hoagland solution containing 0, 100 and 200 mM NaCl. The effects of salt stress on growth, physiological and biochemical parameters were determined after 180, 210, and 240 days of treatment. Saline stress response was evaluated in leaves by measuring the activity of GSH and CAT enzymatic activity, as well as proline levels, gas exchanges, leaves relative water content and chlorophyll content, and proline content. All the studied cultivars showed a decrease in Net Photosynthesis, leaves chlorophyll content and plant growth (mainly leaves dry weight) and an increase in the activity of GSH and CAT. In addition, the reduction of proline content in leaf tissues, induced an alteration of osmotic regulation. Among the studied cultivars Royal and Koroneiki better counteracting the effects of saline stress thanks to a higher activity of two antioxidant enzymes.

Selenium maintains Ca2+ homeostasis in sheep lymphocytes challenged by oxidative stress.

Selenium (Se) is an essential element in human and animal diets, based upon a widespread range of beneficial effects that are primarily due to its antioxidant properties. While Se can be associated to anti-cancer and anti-diabetic activities, reproductive efficiency, and enhancement of the immune system, the mechanistic details of the corresponding biological processes are still largely elusive. To avoid deficiencies and increase bioavailability, Se it is generally supplied to livestock through Se-supplemented feeds or forage plants fertilized with inorganic Se. While the relationship between Ca2+ and ROS (reactive oxygen species) is well known, only a few studies have addressed the possible involvement of Se in the control of cytosolic Ca2+ in oxidative stress. The results on Ca2+ homeostasis were obtained adding exogenous Se in the form of SeO42- to sheep lymphomonocytes cultured in vitro. In particular, Se strongly attenuated 1mM H2O2-induced alteration of intracellular [Ca2+]C as well as the entry of extracellular Ca2+ into the cells with comparable EC50 values for sodium selenate accounting to 1.72 and 2.28 mM, respectively. In an ex vivo trial, it was observed that Ca2+ homeostasis can effectively be rescued in sheep lymphomonocytes exposed in vivo to a Se concentration of approximately 1.9 mM, that was achieved by feeding sheep with olive leaves previously sprayed with 500 mg/plant Na-selenate. Thus the results obtained suggest that the mode of action of selenium markedly influenced Ca2+-related signaling events. Furthermore, results clearly reveal that the protective effect of Se on Ca2+ homeostasis under oxidative challenge can be clearly and effectively achieved through an appropriate dietary regimen obtained also in a circular economy logic using pruning of olive trees treated to reduce tree drought stress.

Trans-crocetin improves amyloid-ß degradation in monocytes from Alzheimer's Disease patients.

Herbal medicines have been recently employed in research and clinical studies for the potential treatment of behavioral and psychological symptoms associated with Alzheimer's Disease (AD) and other types of dementia. The present study investigates the effect of trans-crocetin, an active constituent of Crocus sativus L., to restore in vitro the reduced ability of AD patients' monocytes to degrade amyloid-ß(1-42) (Aß42). CD14+ monocytes from 22 sporadic AD patients with moderate cognitive impairment were isolated; then, the role of trans-crocetin, purified from saffron extracts, was evaluated in terms of Aß42 degradation rate through flow cytometry, as well as expression of cathepsin B by Western blotting. We observed that low micromolar doses of trans-crocetin enhanced Aß42 degradation in AD monocytes through the upregulation of the lysosomal protease cathepsin B. CA074Me, a potent and selective cathepsin B inhibitor, counteracted such trans-crocetin-induced effect. These data suggest that the carotenoid trans-crocetin improves in vitro the clearance of Aß42 through the involvement of cathepsin B, and this could be of value in developing a new anti-amyloid strategy in AD.

Antagonistic Effect of a Salivary Proline-Rich Peptide on the Cytosolic Ca2+ Mobilization Induced by Progesterone in Oral Squamous Cancer Cells.

A salivary proline-rich peptide of 1932 Da showed a dose-dependent antagonistic effect on the cytosolic Ca2+ mobilization induced by progesterone in a tongue squamous carcinoma cell line. Structure-activity studies showed that the activity of the peptide resides in the C-terminal region characterized by a proline stretch flanked by basic residues. Furthermore, lack of activity of the retro-inverso peptide analogue suggested the involvement of stereospecific recognition. Mass spectrometry-based shotgun analysis, combined with Western blotting tests and biochemical data obtained with the Progesterone Receptor Membrane Component 1 (PGRMC1) inhibitor AG205, showed strong evidence that p1932 performs its modulatory action through an interaction with the progesterone receptor PGRMC1, which is predominantly expressed in this cell line and, clearly, plays a role in progesterone induced Ca2+ response. Thus, our results point to p1932 as a modulator of the transduction signal pathway mediated by this protein and, given a well-established involvement of PGRMC1 in tumorigenesis, highlight a possible therapeutic potential of p1932 for the treatment of oral cancer.

Nitric oxide in ischemic and reperfused human muscle.

BACKGROUND: Biochemical events explaining the pathology of ischemia-reperfusion in the muscle are still debated. Nitric oxide (NO) has been postulated to be implicated in these phenomena, but the short half-life of this compound makes it difficult to measure. METHODS: In this paper, we used an amperometric solid-sate sensor to measure NO concentrations in frozen human muscles before, during and after a period of ischemia. We also measured cytochrome oxidase activity and malondialdehyde (MDA). RESULTS: NO increased during ischemia but it soon returned to normal values upon reperfusion. On the other hand, cytochrome oxidase that also decreased in ischemic muscle did not increase during the reperfusion and malondialdehyde only increased during reperfusion, indicating the occurrence of peroxidative reactions in this situation. CONCLUSIONS: NO is implicated in the ischemia/reperfusion pathology, but it is difficult to relate whether this is connected to cytochrome oxidase activity and malondialdehyde formation, also modified in this ischemia-reperfusion model.

The impact of nitric oxide on calcium homeostasis in PE/CA-PJ15 cells.

OBJECTIVE: Nitric oxide (NO) production and Ca(2+) homeostasis are key determinants for the control of many cell functions. NO is known to be a mediator of Ca(2+) homeostasis in a highly complex and cell-specific manner and although Ca(2+) homeostasis has been explored in human oral cancer cells, the exact mechanisms are not completely understood. In this study we investigated the impact of exogenous NO on [Ca(2+)]c homeostasis in PE/CA-PJ15 cells. DESIGN: Cells were treated with S-nitrosocysteine as NO-donor and the determinations of cytosolic Ca(2+) concentrations were performed using FURA-2 AM. Carbonyl cyanide p-(trifluoromethoxy) phenylhydrazone (FCCP) and oligomycin were used to challenge mitochondrial functionality, whereas thapsigargin (TG) and La(3+) were employed to perturb intracellular calcium levels. RESULTS: NO derived from S-nitrosocysteine (CySNO) induced a dose-dependent reduction of cytosolic calcium [Ca(2+)]c whereas oxy-haemoglobin (oxyHb) completely counteracted this effect. Subsequently, we assessed possible relationships between NO and cellular structures responsible for Ca(2+) homeostasis. We found that uncoupling of mitochondrial respiration with carbonyl-cyanide-4-(trifluoromethoxy)-phenylhydrazone (FCCP) and oligomycin strongly reduced the effect of NO on [Ca(2+)]c. Moreover, we found that during this mitochondrial energetic deficit, the effect of NO on [Ca(2+)]c was also reduced in the presence of La(3+) or thapsigargin. CONCLUSIONS: NO induces a concentration-dependent [Ca(2+)]c reduction in PE/CA-PJ15 human oral cancer cells and potentiates mitochondrial Ca(2+) buffering in the presence of TG or La(3+). Further, we show that exogenous NO deregulates Ca(2+) homeostasis in PE/CA-PJ15 cells with fully energized mitochondria.

Formation of nitrosothiols from gaseous nitric oxide at pH 7.4.

Nitric oxide (NO) is generated in biological systems and plays important roles as a regulatory molecule. Its ability to bind to haem iron is well known. Moreover, it may lose an electron, forming the nitrosonium ion, involved in the synthesis of S-nitrosothiols (SNOs). It has been suggested that S-nitrosohaemoglobin (-SNO Hb) and low molecular weight SNOs may act as reservoirs of NO. SNOs are formed in vitro, at strongly acidic pH values; however, the mechanism of their formation at neutral pH values is still debated. In this paper we report the anaerobic formation of SNOs (both high- and low-molecular weight) from low concentrations of NO at pH 7.4, provided Hb is also present. We propose a reaction mechanism entailing the participation of Fehaem in the formation of NO(+) and the transfer of NO(+) either to Cysbeta(93) of Hb or to glutathione; we show that this reaction also occurs in human RBCs.

Nitric oxide and fusion with prostasomes increase cytosolic calcium in progesterone-stimulated sperm.

Spermatozoa must undergo a number of reactions before they are able to fertilize the oocyte. Among these is the acrosome reaction, which is related to an increase in cytosolic Ca2+ concentration ([Ca2+]i). It has been reported in the literature that progesterone may achieve this effect through the intervention of extragenomic receptors. Nitric oxide (NO) has been reported to affect spermatozoa; the nature of the effect depends on the concentration of the radical. In a previous paper, we reported that the fusion of spermatozoa with prostasomes may also produce a transient increase in spermatozoa [Ca2+]i; in addition, this phenomenon causes a long-lasting effect that influences the action of progesterone. In this paper, we test the effects of a NO donor (CysNO) and of fusion of the prostasome to spermatozoa on progesterone-induced [Ca2+]i increase. No effect at all was noticed in the absence of progesterone stimulation. In the presence of the hormone, both CysNO and fusion increased the progesterone effect. This phenomenon was much more evident if the two treatments were used together. We conclude that both NO and fusion with prostasomes act on the progesterone-dependent pathway additively. Probably the effects are independent.