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Supplementary Figures 1-6 from Concomitant Tumor and Minor Histocompatibility Antigen–Specific Immunity Initiate Rejection and Maintain Remission from Established Spontaneous Solid Tumors

CD4+CD25+Foxp3+ regulatory T cells (Treg) are thought to suppress the natural and vaccine-induced immune response against tumor-associated antigens (TAA). Here, we show that Treg accumulate in tumors and tumor-draining lymph nodes of aging transgenic adenocarcinoma of the mouse prostate (TRAMP) male mice, which spontaneously develop prostate cancer. TAA overexpression and disease progression associate also with induction of TAA-specific tolerance. TAA-specific T cells were found in the lymphoid organs of tumor-bearing mice. However, they had lost the ability to release IFN-γ and kill relevant targets. Neither in vivo depletion of Treg by PC61 monoclonal antibody followed by repeated vaccinations with antigen-pulsed dendritic cells nor the combined treatment with 1-methyl-l-tryptophan inhibitor of the enzyme indoleamine 2,3-dyoxigenase, PC61 antibody, and dendritic cell vaccination restored the TAA-specific immune response. Treg did not seem to control the early phases of tolerance induction, as well. Indeed, depletion of Treg, starting at week 6, the age at which TRAMP mice are not yet tolerant, and prolonged up to week 12, did not avoid tolerance induction. A similar accumulation of Treg was found in the lymph nodes draining the site of dendritic cell vaccination both in TRAMP and wild-type animals. Hence, we conclude that Treg accrual is a phenomenon common to the sites of an ongoing immune response, and in TRAMP mice in particular, Treg are dispensable for induction of tumor-specific tolerance. [Cancer Res 2008;68(1):292–300]

Supplementary Figures S1-S7 from Peripheral T-Cell Tolerance Associated with Prostate Cancer Is Independent from CD4+CD25+ Regulatory T Cells

Nonmyeloablative hematopoietic cell transplantation can cure patients with hematologic malignancies but has reported limited success against solid tumors. This is possibly because of profound peripheral tolerance mechanisms and/or suboptimal tumor recognition by effector T lymphocytes. We report that in mice developing spontaneous prostate cancer, nonmyeloablative minor histocompatibility mismatched hematopoietic stem cell transplantation, and donor lymphocyte infusion of unmanipulated lymphocytes combined with posttransplant tumor-specific vaccination circumvents tumor-specific tolerance, allowing acute tumor rejection and the establishment of protective immunosurveillance. Although donor-derived tumor-specific T cells readily differentiated into effector cells and infiltrated the tumor soon after infusion, they were alone insufficient for tumor eradication, which instead required the concomitance of minor histocompatibiltiy antigen–specific CD8+ T-cell responses. The establishment of protective immunosurveillance was best induced by posttransplant tumor-specific vaccination. Hence, these results provide the proof of principle that tumor-specific T-cell responses have to be harnessed together with minor histocompatibility responses and sustained by posttransplant tumor-specific vaccination to improve the efficacy of allotransplantion for the cure of solid tumors. Cancer Res; 70(9); 3505–14. ©2010 AACR.

Objective Fcγ receptors (FcγR) play important roles in both protective and pathogenic immune responses. The assembly of the CBM signalosome encompassing caspase recruitment domain-containing protein 9, B cell CLL/lymphoma 10, and mucosa-associated lymphoid tissue lymphoma translocation protein 1 (MALT-1) is required for optimal FcγR-induced canonical NF-κB activation and proinflammatory cytokine release. This study was undertaken to clarify the relevance of MALT-1 protease activity in FcγR-driven events and evaluate the therapeutic potential of selective MALT-1 protease inhibitors in FcγR-mediated diseases. Methods Using genetic and pharmacologic disruption of MALT-1 scaffolding and enzymatic activity, we assessed the relevance of MALT-1 function in murine and human primary myeloid cells upon stimulation with immune complexes (ICs) and in murine models of autoantibody-driven arthritis and immune thrombocytopenic purpura (ITP). Results MALT-1 protease function is essential for optimal FcγR-induced production of proinflammatory cytokines by various murine and human myeloid cells stimulated with ICs. In contrast, MALT-1 protease inhibition did not affect the Syk-dependent, FcγR-mediated production of reactive oxygen species or leukotriene B4 . Notably, pharmacologic MALT-1 protease inhibition in vivo reduced joint inflammation in the murine K/BxN serum-induced arthritis model (mean area under the curve for paw swelling of 45.42% versus 100% in control mice; P = 0.0007) but did not affect platelet depletion in a passive model of ITP. Conclusion Our findings indicate a specific contribution of MALT-1 protease activity to FcγR-mediated events and suggest that MALT-1 protease inhibitors have therapeutic potential in a subset of FcγR-driven inflammatory disorders.

Tomato (Lycopersicon esculentum Mill. cv. Pearson) plants were grown in growth chambers for 25 days with cadmium (Cd) and then exposed briefly to ozone (O3). Gas exchange, chlorophyll a fluorescence, and pigment composition were analysed in leaves at the end of the treatment to assess the effects of a single pollutant and their combination on photosynthesis. The CO2 assimilation rate was dramatically reduced in plants subjected to the combined treatment, while the single effect of Cd appeared less severe than that of O3. The decline of CO2 photoassimilation found in all O3-exposed plants was attributed to both stomatal and nonstomatal limitations. Tomato plants seemed to detoxify Cd to a great extent, but this resulted in growth suppression. In response to O3 exposure, the plants protected their photosystems by heat dissipation of excess energy via the xanthophyll cycle. Cd combined with O3 affected adversely this cycle resulting in an increase in photosynthetic performance under the same experimental light conditions.

BACKGROUND Polyphenol oxidase (PPO) and, to a minor extent, peroxidase (POD) represent the key enzymes involved in enzymatic browning, a negative process induced by cutting fresh-cut produce such as lettuce (Lactuca sativa) and rocket salad (Eruca sativa). Although ascorbic acid is frequently utilised as an anti-browning agent, its mechanism in the prevention of the browning phenomenon is not clearly understood. RESULTS The activity of PPO and POD and their isoforms in lettuce (a high-browning and low-ascorbic acid species) and rocket salad (a low-browning and high-ascorbic species) was characterised. The kinetic parameters of PPO and in vitro ascorbic acid–PPO inhibition were also investigated. In rocket salad, PPO activity was much lower than that in lettuce and cutting induced an increase in PPO activity only in lettuce. Exogenous ascorbic acid (5 mmol L−1) reduced PPO activity by about 90% in lettuce. POD did not appear to be closely related to browning in lettuce. CONCLUSION PPO is the main enzyme involved in the browning phenomenon; POD appears to play a minor role. The concentration of endogenous ascorbic acid in rocket salad was related to its low-browning sensitivity after cutting. In lettuce, the addition of ascorbic acid directly inhibited PPO activity. The results suggest that the high ascorbic acid content found in rocket salad plays an effective role in reducing PPO activity. © 2012 Society of Chemical Industry

Basil (Ocimum basilicum L., cultivar Genovese) plants were grown in Hoagland solution with or without 50 mM NaCl or 25 mM Na2SO4. After 15 days of treatment, Na2SO4 slowed growth of plants as indicated by root, stem and leaf dry weight, root length, shoot height and leaf area, and the effects were major of those induced by NaCl. Photosynthetic response was decreased more by chloride salinity than by sulphate. No effects in both treatments on leaf chlorophyll content, maximal efficiency of PSII photochemistry (Fv/Fm) and electron transport rate (ETR) were recorded. Therefore, an excess of energy following the limitation to CO2 photoassimilation and a down regulation of PSII photochemistry was monitored under NaCl, which displays mechanisms that play a role in avoiding PSII photodamage able to dissipate this excess energy. Ionic composition (Na+, K+, Ca2+, and Mg2+) was affected to the same extent under both types of salinity, thus together with an increase in leaves Cl−, and roots SO42− in NaCl and Na2SO4-treated plants, respectively, may have resulted in the observed growth retardation (for Na2SO4 treatment) and photosynthesis activity inhibition (for NaCl treatment), suggesting that those effects seem to have been due to the anionic component of the salts.

The aim of the present work was to evaluate the effects of water stress and different rootstocks on the quality indices and nutritional characteristics of peach fruit, cv. Suncrest. The results show how pre-harvest factors, such as the type of rootstock and water stress, can influence the quality indices and nutritional properties of peach fruits. Rootstocks type influenced both quality indices such as flesh firmness (FF) and soluble solid content (SSC), but it is unclear how rootstock exerted its influence. Also water stress induced changes in quality indices of peach fruit grafted on GF 677 and Montclar whereas minor effects were recorded when water stress was applied on Penta. The content of hydroxycinnamic acids was similar in control fruits of GF677, Montclar and Penta and it increased significantly only in fruit of trees grafted on GF 677 following water stress. Anthocyanins content in fruit of control plants was higher in Montclar in comparison with the fruit grafted on the other two rootstocks while procyanidin content was higher in fruit of control plants grafted on Penta. Following the water stress an increase in total anthocyanins was observed in GF 677 and Penta whereas no significant differences were found for Montclar. Water stress induced a significant decrease in procyanidin content in Penta. In conclusion in fruits of trees grafted on Penta following water stress quality indices did not show changes but significant variations in some phytochemicals, i.e. anthocyanins and procyanindins were observed. The results suggest that the regulation of water management is an important factor to maintain a good quality of the fruits. In fact, if irrigation stress can induce a higher biosynthesis of phytochemical compounds and an increase of SSC, at the same time, it can change fruit quality traits, such as a lower FW in Montclar, a higher titratable acidity (TA) in GF 677 and Penta and a lower FF in Gf 677 and Montclar.

Despite the remarkable morphological diversity in the leaf shape, usually higher plants maintain a flat lamina to ensure the desirable surface-to-volume ratio. The control of expansion of a flattened lateral organ is only partially known and the characterization of leaf mutants can be a useful strategy to add new information, as demonstrated in dicots with recessive mutants of model species. In sunflower, the dominant mutant Basilicum Leaf (BL) is characterized by prominent downward leaf curling. The central end of the present work is the detailed study of the BL phenotype to characterize the effects of this mutation, at organ and cellular levels, on both morphological and physiological traits. The geometry of mature leaves is obviously altered in BL plants because the lamina surfaces are downward curled and display a different length/width ratio with respect to wild type. The area of BL leaves is higher than that of wild type while the BL leaf perimeter is reduced. In BL leaves, the perimeter to the square root of area ratio is also abnormally low compared to the value of a flat sunflower lamina. These results suggest that the dominant mutation induces a deep change of the ratio between lamina and marginal growth. In addition, the epidermis and mesophyll of BL leaves comprised higher size pavement and palisade cells compared to those of wild type, indicating that the BL gene also control cell shape changes in the formation of the leaf lamina. Other leaf traits such as photosynthetic pigment content, pattern of minor vein architecture, margin type and stomatal density are affected by the mutation. In BL plants, the longitudinal axis of both cotyledons and ray flower corollas shows an unusual curvature while the diameter of shoot and inflorescence meristems is reduced. In mutant leaves, the gas exchange performance is influenced by low stomata conductance while the CO2 assimilation rate is also depressed by a depleted Rubisco activity. Together, these data demonstrate the pleiotropic nature of the BL mutation, which affects a number of morphological aspects of flattened lateral organs as well as physiological parameters in sunflower.

Two bean cultivars with different sensitivity to ozone, i.e. the O 3 -sensitive Cannellino and the O 3 -tolerant Top Crop, were exposed to acute O 3 -stress (165 nL L −1 ) with the aim of evaluating physiological and biochemical traits that may confer O 3 -tolerance. Stomatal conductance was smaller and the ability to dissipate excess energy, via regulated and unregulated nonphotochemical quenching mechanisms was greater in Top Crop than in Cannellino. These morphological and physiological-traits allowed the O 3 -tolerant cultivar to compensate for the light-induced declines in Φ PSII , to preserve photosystem II from excitation-energy, and likely to prevent the generation of ROS to a superior degree than the O 3 -sensitive cultivar. Furthermore, the potential capacities to reducing the superoxide anion and H 2 O 2 were significantly greater in Top Crop than in Cannellino. These findings are consistent with the early accumulation of H 2 O 2 , the almost complete disruption of cell structure, and irreversible damages to the photosynthetic apparatus observed in the O 3 -sensitive cultivar.

Softening process and total antioxidant activity were evaluated in kiwifruit differently exposed to light intensity and harvested at two different times (October 10 and November 14). Fruit was stored for 2 months at 0 °C (S1) and then maintained for a week at ambient temperature (S2). The results showed that fruit harvested later presented a faster softening rate during storage than fruit harvested earlier, even if antioxidant activity did not change. Light-exposed fruit showed higher flesh firmness than that of shaded ones. Polygalacturonase activity was higher in kiwifruit maintained for a week at ambient temperature after cool storage and, in particular the highest value was recorded in fruit harvested later. The behaviour of β-galactosidase was different: it did not show changes in fruit harvested later and significantly decreased in light- and shade-exposed fruit harvested earlier.

Root-zone salinity stress and high solar irradiance concomitantly occurs in the Mediterranean basin, where Olea europaea is the dominating fruit-tree crop-species. Although the effect of each individual stressor on plant performance has been widely investigated, much less is known on the interaction effects of salinity stress and solar irradiance on the physiology and biochemistry of olive plants. Here we analyzed how changes in root-zone NaCl concentration and sunlight radiation affect relevant physiological and biochemical features in olive cv. Cipressino. Two-year-old plants were supplied with 0 or 125 mM NaCl and exposed to 15% (shade) or 100% sunlight (sun) over a 5-week period, starting from July 10th, 2005. Measurements were conducted of (i) gas exchange and plant growth, (ii) the concentrations of cations and chloride, (iii) the concentrations of soluble carbohydrates, violaxanthin-cycle pigments and polyphenols, and (iv) the protein oxidation and the lipid peroxidation in the leaves. Salt-induced reductions in gas exchange performance and plant growth were greater at the sun than at the shade site, mostly due to light-induced changes in leaf water relations and vapour pressure deficit (vpd), rather than in the concentration of potentially toxic ions. Light-induced increases in leaf Na + and Cl − concentrations were countered by parallel enhancements in the concentrations of K + and Ca 2+ . Sun leaves had sharply greater concentrations of mannitol and xanthophylls, irrespective of root-zone salinity. The amount of “newly assimilate carbon” allocated to polyphenols, especially to flavonoids, increased in response to salinity stress and high sunlight. Remarkably, the protein oxidation was greater in shade than in sun leaves of well-watered plants, and increased more at the shade than at the sun site because of high salinity. We suggest that heat-stress (on average maximum T exceeded 33 °C for 50% of the experimental period), which acted in concert with salinity stress and sunlight irradiance in determining plant responses in our experiment, was responsible for leaf oxidative damage in plants growing under contrasting solar radiation. Indeed, sun leaves of salt-stressed plants were equipped with an extraordinary-rich arsenal of antioxidant compounds, distributed in different cell compartments, i.e., mannitol, zeaxanthin and flavonoids, which likely countered effectively the oxidative damage driven by heat-stress, a clear example of cross-tolerance.

In this study the response to photoinhibition of photosynthesis and subsequent recovery was examined in plants of Phaseolus vulgaris L. cultivar 'Pinto' exposed to charcoal-filtered air or to ozone (O-3) at 150 nL L-1 either for 3 h, or for 5 h. The responses were analysed using chlorophyll fluorescence imaging and by conventional fluorometry. Compared to control plants maintained in charcoal-filtered air, in plants exposed for 3 h to O-3 and then subjected to high light treatment, the results show an increased tolerance to photoinhibition. Plants exposed to the same O-3 concentration but for the longer 5-h period, were not tolerant to the photoinhibition treatment and, instead showed visible symptoms of damage (chlorosis and necrosis) clearly attributable to the longer O-3 exposure. Here the detrimental effects of O-3 aggravated the effects of the high light photoinhibitory treatment. The leaves exposed to the shorter O-3 treatment (150 nL L-1 for 3 h) developed an ability to counteract the negative effects of a high light exposure probably because the O-3 had activated an antioxidant system able to protect the photosynthetic machinery. (C) 2008 Elsevier Ireland Ltd. All rights reserved.

In this study, the effect of nitrogen form (nitrate and ammonium in the molar ratio of 1:1 or 1:0), supplied to the plant or NaCl salinity (up to a concentration of 50 mol m−3) of the feeding solution, on the content of selected caffeic acid derivatives and the activities of phenylalanine ammonia lyase (PAL; EC. 4.3.1.5) and shikimate dehydrogenase (SKDH; EC 1.1.1.25) were investigated in the leaves and the roots of Echinacea angustifolia DC, grown hydroponically. Two experiments were conducted under glasshouse conditions with seedlings transplanted in aerated stagnant nutrient solution (floating raft system) four weeks after emergence. The nitrate/ammonium ratio in the nutrient solution did not affect plant growth or dry matter partitioning, but it influenced the concentration of determined metabolites; in particular, the root concentrations of chlorogenic acid, echinacoside and caffeic acid were significantly higher in the plants grown with nitrate as sole source of nitrogen. NaCl salinity reduced plant growth, by reducing the biomass allocated to the inflorescences, and enhanced the accumulation of chlorogenic acid and cichoric acids, at least in the roots.

The influences of harvest time and storage on the quality indices and nutritional content of kiwifruit were evaluated. Antioxidant capacity, ascorbic acid, total phenol content, carotenoids, soluble solids content and flesh firmness were determined in kiwifruit gathered at two different time (T1: 17-11-2005 and T2: 24-11-2005) and stored at 0 °C, for 2 or 6 months (S1 and S2, respectively). At the end of the cool storage, fruits were maintained for a week at 25 °C (S1 + 7d and S2 + 7d). The flesh firmness was reduced at the end of cool storage and the soluble solids content significantly increased, for exception of fruits harvested at T2 and stored for 6 months at 0 °C and a week at ambient temperature (S2 + 7d). Some nutritional characteristics such as vitamin C and carotenoids were higher in fruits gathered at T1 but these parameters were strongly influenced by storage, with a general decrease at the end of the long cool storage (6 months). Differently, no influence of long storage was observed in the fruit collected at T2 time. The maintenance for a week at room temperature, after long cool storage, determined an improvement of nutritional characteristics of kiwifruits. In conclusion, fruits harvested at T2 seem to improve their quality after a long storage (6 months) because they reach nutritional values similar or higher than those recorded in fruits at the harvest time. In spite of these positive results, these fruits showed a reduction in organoleptic characteristics which could negatively influence the fruit marketing. The obtained results underline the important role of the pre- and post-harvest factors on the qualitative and nutritional characteristics of kiwifruits.

The browning process was studied in two cultivars of minimally processed lettuce (Lactuca sativa var. capitata cv. Verpia and Lactuca sativa var. acephala cv. Lollo Rossa). The analyses were carried out upon storage at 4 °C in the dark. The first interesting result was the resistance to browning of excised leaves of Lollo Rossa, that did not present the typical symptoms of browning. Another important characteristic of Lollo Rossa lettuce was the higher antioxidant ability before storage. The results of biochemical characteristics obtained in this experiment did not show clear evidence supporting the biochemical model of tissue browning involving the sequential synthesis of phenolics and quinones that are regulated, respectively, by phenyl alanine ammonia-lyase, polyphenol oxidase and/or peroxidase.

In fresh-cut leafy vegetables, the operation of cutting may stimulate enzymatic browning, with important commercial consequences. In this work, a number of physiological and biochemical parameters, including the activities of key enzymes involved in the metabolism of phenols (such as PAL, PPO, and PODs) and ascorbic acid (ASA), were measured in three species: lettuce (Lactuca sativa var. capitata L.), escarole (Cichorium indivia var. latifolium) and rocket salad (Eruca sativa), upon cold storage as fresh cuts. The first two species are quite sensitive to leaf browning, which does not affect rocket salad. The resistance of rocket salad to browning was associated with a much higher ASA content and a decrease in this compound upon storage, compared to other species in which ASA remained either constant (lettuce) or increased (escarole). It is proposed that the resistance of rocket salad to this disorder, which markedly affects other leafy species, e.g., lettuce and escarole, is a result of the inhibition of PPO activity and/or the reduction of quinones to phenols, which may both be induced by ASA.

Dendritic cell (DC) maturation influences the priming and polarization of T lymphocytes. We recently found that early activated DC (i.e. DC exposed to pro-maturation stimuli for 8 h) were more prone to prime in vivo a type-1 cytotoxic T cell (Tc1) response than DC exposed to pro-maturation stimuli for 48 h (48h-DC). We investigated whether 48h-DC, conversely, allowed the induction of Tc2 cells. Antigen-pulsed mouse bone-marrow-derived DC at any maturation stage, in the presence of exogenous IL-12, skewed in vitro naive CD8(+) T cells towards Tc1 cells, but 48h-DC most potently, in the presence of exogenous IL-4, favored the induction of Tc2 cells. In vivo, full maturation of DC promoted expansion of Tc2 and fall of Tc1 cells. Tc2 cells maintained a high cytolytic activity and produced significant amounts of IL-4, IL-5, IL-10 and TGF-beta. Our results indicate that polarization of naive CD8(+) T cells to Tc2 cells is dependent on the amount of time DC have been exposed to maturation stimuli, and might be favored in late and/or chronic phases of an immune response.

Activated CD8+ T cells can differentiate into type 1 (Tc1) cells, producing mainly IFN-γ, and type 2 (Tc2) cells, producing mostly IL-4, IL-5, and IL-10. Tc1 cells are potent CTL involved in the defense against intracellular pathogens and cancer cells. The role of Tc2 cells in the immune response is largely unknown, although their presence in chronic infections, cancer, and autoimmune diseases is associated with disease severity and progression. Here, we show that mouse Tc2 cells modify, through a cell-to-cell contact mechanism, the function of bone marrow-derived dendritic cells (DC). Indeed, Tc2-conditioned DC displayed a reduced expression of MHC class II and costimulatory molecules, produced IL-10 instead of IL-12, and favored the differentiation of both naive CD4+ and CD8+ T cells toward type 2 cells in the absence of added polarizing cytokines. The novel function for Tc2 cells suggests a type 2 loop in which Tc2 cells modify DC function and favor differentiation of naive T cells to type 2 cells. The type 2 loop may at least in part explain the unexpected high frequency of type 2 cells during a chronic exposure to the Ag.

In the tumor-prone transgenic adenocarcinoma mouse prostate (TRAMP) mouse model we followed the fate of the immune response against the SV40 large T antigen (Tag) selectively expressed in the prostate epithelium during the endogenous transformation from normal cells to tumors. Young (5-7-week-old) male TRAMP mice, despite a dim and patchy expression of Tag overlapping foci of mouse prostate intraepithelial neoplasia, displayed a strong Tag-specific cytotoxic T lymphocyte (CTL) response after an intradermal injection of peptide-pulsed dendritic cells (DC). This response was weaker than the one found in vaccinated wild-type littermates, and was characterized by a reduced frequency and avidity of Tag-specific CTL. Early DC vaccination also subverted the profound state of peripheral tolerance typically found in TRAMP mice older than 9-10 weeks. The DC-induced CTL response indeed was still detectable in TRAMP mice of 16 weeks, and was associated with histology evidence of reduced disease progression. Our findings suggest that tumor antigens are handled as self antigens, and peripheral tolerance is associated with in situ antigen overexpression and cancer progression. Our data also support a relevant role for DC-based vaccines in controlling the induction of peripheral tolerance to tumor antigens.

The current study evaluated photosynthetic processes in two clover species based on their performance under high O3 conditions (150 nl l ‐1 for 3 h). These species are Trifolium repensL. and Trifolium pratenseL., which are well known for their different sensitivity to ozone. Ozone affected the two clover species very differently. In T. pratense, ozone induced visible symptoms of damage even though CO2 photoassimilation, stomatal conductance and the electron transport rate were not affected. A decrease in the optimal quantum yield was observed in T. pratense immediately after the end of the period of O3 stress but it reverted to a value similar to the control 24 h after removing the stress, indicating that non-irreversible photoinhibition had occurred. Data obtained for symptomatic T. pratense indicate that still-green living tissue of the leaf is able to carry out CO2 assimilation; the only parameter found to be affected by O3 was the efficiency of excitation capture. In T. repens, acute O3 fumigation induced inhibition of photosynthetic activity, enhanced stomatal closure and increased the reduction state of the PSII primary acceptor. A possible explanation for the inhibition of photosynthesis could reside in inhibition of the Calvin cycle over-reducing PSII, thus increasing (1 ‐qP) and increasing non-photochemical quenching. © 2003 Editions scientifiques et medicales Elsevier SAS. All rights reserved.

Summary • Bean seedlings (Phaseolus vulgaris cv. Pinto) were grown in the greenhouse at a light intensity of 400 µmol m−2 s−1. When the primary leaf was fully expanded, plants were divided into four groups and subjected to one of the following treatments: light intensity of 400 µmol m−2 s−1 and filtered air (control); light intensity of 400 µmol m−2 s−1 and ozone (O3) (150 nl l−1 for 5 h) (ozonated); light intensity of 1000 µmol m−2 s−1 for 5 h and filtered air (HL); and light intensity of 1000 µmol m−2 s−1 and O3 (150 nl l−1) for 5 h (HL + O3). • At the end of the treatments (HL and/or O3) a strong decrease in CO2 assimilation rate as well a decrease in stomatal conductance were observed, while no changes in intercellular CO2 concentration were recorded. In addition the Fv : Fm ratio (maximal quantum yield for PSII photochemistry) decreased in the stressed leaves (HL and/or O3), indicating photoinhibition, and they showed a corresponding increase in minimal fluorescence (F0), indicating a higher number of deactivating photosystem II (PSII) centres. • The maximum catalytic activity of the Benson–Calvin cycle enzymes, fructose-1,6-bisphosphate phosphatase (FBPase) and Rubisco, decreased following HL + O3 stress but activation was enhanced. A linear relation was found between activation state of NADP-malate dehydrogenase (MDH) and the flux of electrons through PSII and in HL + O3-treated plants NADP-MDH activity decreased at high irradiance levels, indicating a limitation in linear electron flux.

The effects of long-term exposure to ozone (O3, 60 mm3 m-3 for 5 h d-1) on some Calvin cycle enzymes, in particular those modulated by the thioredoxin system, were studied in two poplar clones. These clones differ in sensitivity to O3. In the I-214 clone, the first effects from O3 treatment were seen after 40 d of fumigation, while the Eridano clone showed visible symptoms of damage after only 15 d of the treatment. Specific activity of ribulose-1,5-bisphosphate carboxylase/oxygenase (E.C. 4.1.1.39) diminished in both the clones, while specific activity of phosphoenolpyruvate carboxylase (E.C. 4.1.1.31) increased. Exposure to O3 also caused a reduction in the specific activity of ribulose-1,5-bisphosphate kinase (E.C. 2.7.1.19) in both clones. At the end of the exposure to O3, specific activity of glyceraldehyde 3-phosphate dehydrogenase (E.C. 1.2.1.13) increased in I-214 and remained similar to the control in Eridano, whereas specific activity of fructose-1,6-bisphosphate phosphatase (E.C. 3.1.3.11) was higher in Eridano and similar to the control in I-214.

Iron (Fe) is an essential element for plants and its deficiency causes decrease not only in the photosynthetic rate but also in the actual photosystem II efficiency at steady-state photosynthesis. The aim of this work was to determine the effect of Fe deficiency in plants of Cucumis sativus (L.) in two different experimental conditions. In the first experiment, plants were grown with or without Fe for 7 d. After 7 d, Fe-deficient plants were resupplied with Fe and sampled after 12 h and 48 h. In the second experiment, plants were grown with Fe in the nutrient solution for 3 d and after this period, Fe was withdrawn and plants sampled after 3 and 6 d. Iron and chlorophyll (Chl) concentration and Chl-fluorescence imaging were measured. In cucumber leaves subjected to Fe deficiency, fluorescence imaging of Chl a evidenced spatial changes on leaf lamina. Following Fe deficiency both after 7 d (Exp. 1) or 6 d (Exp. 2) leaves showed a slight, nonsignificant decrease in Fv/Fm ratio. However Chl-fluorescence parameters determined in light conditions showed significant changes which indicate an alteration in the photosynthetic process. Surprisingly, the effect of Fe deficiency was more pronounced in leaves of plant of Exp. 2 as compared to those that had grown in complete absence of Fe (Exp. 1). In the latter case down-regulated mechanisms preserved leaves from irreversible photoinhibition leading to complete recovery when plants were resupplied with the microelement.

Polyphenol oxidase (PPO) and, to a minor extent, peroxidase (POD) represent the key enzymes involved in enzymatic browning, a negative process induced by cutting fresh-cut produce such as lettuce (Lactuca sativa) and rocket salad (Eruca sativa). Although ascorbic acid is frequently utilised as an anti-browning agent, its mechanism in the prevention of the browning phenomenon is not clearly understood.The activity of PPO and POD and their isoforms in lettuce (a high-browning and low-ascorbic acid species) and rocket salad (a low-browning and high-ascorbic species) was characterised. The kinetic parameters of PPO and in vitro ascorbic acid-PPO inhibition were also investigated. In rocket salad, PPO activity was much lower than that in lettuce and cutting induced an increase in PPO activity only in lettuce. Exogenous ascorbic acid (5 mmol L(-1)) reduced PPO activity by about 90% in lettuce. POD did not appear to be closely related to browning in lettuce.PPO is the main enzyme involved in the browning phenomenon; POD appears to play a minor role. The concentration of endogenous ascorbic acid in rocket salad was related to its low-browning sensitivity after cutting. In lettuce, the addition of ascorbic acid directly inhibited PPO activity. The results suggest that the high ascorbic acid content found in rocket salad plays an effective role in reducing PPO activity.

Boron (B) is a micronutrient that has long been kno wn essential not only for plants and animals. Other wise, in crop plants the range of B concentration from es sentiality to toxicity appears extremely narrow. Al though B deficiency has been well investigated, during rec ent years many researchers have also investigated b oron toxicity effects, making progress in the comprehens ion of boron transport within plants. Moreover, worldwide B is not uniformed distributed and, espec ially in arid and semiarid regions, high B concentrations are found in the soil. Boron in plan ts primarily moves following transpiration stream e ven though recent evidences have also confirmed the inv olvement of B transporter and channel (BOR and NIP). In general, the species that tolerate B excess tend s to accumulate less B compared to sensitive specie s. Likewise other abiotic stresses, antioxidants may b e involved in the scavenging of Reactive Oxygen Species (ROS) triggered by B toxicity. Photosynthesis parameters in plants grown under high B supply can draw the photosystems situation and the reactions to B toxic ity drawing the healthy status of the plants evalua ted. Although boron deficiency has been well investigate d, during recent years many researchers have also investigated boron toxicity effects, making progres s in the comprehension of boron transport within pl ants. This review represents the actual state of knowledg e concerning B toxicity.

Chlorophyll (Chl) fluorescence is a non-invasive technique employed to measure photosynthesis in leaves, widely used to study plant’s response to abiotic stresses. In fact, Chl fluorescence parameters can be efficiently used to evaluate changes in photosystem II photochemistry, linear electron transport rate and CO2 assimilation in vivo. In this paper also the role of photochemical and non-photochemical quenching involved in the changes in PSII quantum efficiency are discussed. In addition to, it is now practical to use Chl fluorescence imaging to detect heterogeneous patterns of photosynthetic performance across leaves. It is known that photosynthetic apparatus is the main target of many abiotic stresses being chloroplast the organelle which posses pigments that absorb light and drive redox reactions of thylakoids but also the site in the cell where O2 is evolved from water. Clearly, the availability of O2 near of the electron transport chain may lead to the formation of reactive oxygen species (ROS) in the cellular environment. For this reason this paper reports how Chl fluorescence has been conveniently used to detect the responses of plants to abiotic stresses such as ozone, drought, temperature, light intensity, UV radiation and salinity.

Imaging chlorophyll fluorescence was used to study the response of greenhouse tomato grown in soilless culture to NaCl and B concentration in the irrigation water. The plants were grown in perlite with recycling nutrient solution, which was prepared with negligible concentration or 550 mg L−1 of NaCl, and 0.5 or 2.0 mg L−1 of B in the irrigation water. High B concentration resulted in evident necrosis of leaf margins. At high NaCl concentration the recycling nutrient solution was replaced more frequently than at low salinity and this resulted in lower B concentration in the root zone, thus limiting the incidence of necrotic areas in the leaves. Indeed, the severity of leaf damage was linearly related to the mean B concentration in the recycling water. In all treatments we observed a strong difference in B concentrations between marginal and central areas, the latter remaining green and viable even in condition of severe B toxicity, as indicated by the determination of chlorophyll fluorescence. A reduction of the photosynthetic activity was observed in the symptomatic areas of the leaves on the plants grown at high B concentration in the root zone as a result of reduced dissipation of the excess of excitation energy at PSII level.

Chlorophyll (Chl) fluorescence is a tool which is widely used to examine photosynthetic performance in algae and plants. It is a non-invasive analysis that permits to assess photosynthetic performance in vivo (Baker, 2008; Baker & Rosenqvist, 2004; Chaerle & Van Der Straeten, 2001; Woo et al. 2008). Chl fluorescence analysis is widely used to estimate photosystem II (PSII) activity, which is an important target of abiotic stresses (Balachandran et al., 1994; Baker et al., 1983; Briantais et al., 1996; Calatayud et al., 2008; Chaerle & Van Der Straeten, 2000; Ehlert & Hincha, 2008; Gilmore & Govindjee, 1999; Guidi et al., 2007; Guidi & Degl’Innocenti, 2008; Hogewoning & Harbinson, 2007; Krause, 1988; Lichtenthaler et al., 2007; Massacci et al., 2008; Osmond et al., 1999; Scholes & Rolfe, 1996; Strand & Oquist, 1985). It is know as the energy absorbed by Chl molecules must be dissipated into three mechanisms, namely internal conversion, fluorescence and photochemistry (Butler, 1978). All of these downward processes competitively contribute to the decay of the Chl excited state and, consequently, an increase in the rate of one of these processes would increase its share of the decay process and lower the fluorescence yield. Typically, all processes that lower the Chl fluorescence yield are defined with the term quenching. Kaustky and co-workers (1960) were the first which observed changes in yield of Chl fluorescence. These researchers found that transferring a leaves from the dark into the light, an increase in Chl fluorescence yield occurred. This increase has been explained with the reduction of electron acceptors of the PSII and, in particular, plastoquinone QA: once PSII light harvesting system (LCHII) absorbs light and the charge separation occurs, QA accepts electron and it is not able to accept another electron until it has been passed the first one onto the subsequent carrier, namely plastoquinone QB. During this time the reaction centers are said to be closed. The presence of closed reaction centers determines a reduction in the efficiency of PSII photochemistry and, consequently, an increase in the Chl fluorescence yield. Transferring the leaf from the dark into light, PSII reaction centers are progressively closed, but, following this time, Chl fluorescence level typically decreases again and this phenomenon is due to two types of quenching mechanisms. The presence of light induced the activation of enzymes involved in CO2 assimilation and the stomatal aperture that determines that electrons are transferred away PSII. This induced the so-called photochemical quenching, qP. At the same time, there is an increase in the conversion of light energy into

Nonmyeloablative hematopoietic cell transplantation can cure patients with hematologic malignancies but has reported limited success against solid tumors. This is possibly because of profound peripheral tolerance mechanisms and/or suboptimal tumor recognition by effector T lymphocytes. We report that in mice developing spontaneous prostate cancer, nonmyeloablative minor histocompatibility mismatched hematopoietic stem cell transplantation, and donor lymphocyte infusion of unmanipulated lymphocytes combined with posttransplant tumor-specific vaccination circumvents tumor-specific tolerance, allowing acute tumor rejection and the establishment of protective immunosurveillance. Although donor-derived tumor-specific T cells readily differentiated into effector cells and infiltrated the tumor soon after infusion, they were alone insufficient for tumor eradication, which instead required the concomitance of minor histocompatibiltiy antigen–specific CD8+ T-cell responses. The establishment of protective immunosurveillance was best induced by posttransplant tumor-specific vaccination. Hence, these results provide the proof of principle that tumor-specific T-cell responses have to be harnessed together with minor histocompatibility responses and sustained by posttransplant tumor-specific vaccination to improve the efficacy of allotransplantion for the cure of solid tumors. Cancer Res; 70(9); 3505–14. ©2010 AACR.

We have conducted a UV-exclusion experiment in which plants grew at 35 or 100% sunlight irradiance, in the presence or in the absence of UV-irradiance, with the aim of exploring the effects of visible-light and UV-irradiance on the physiology and biochemistry of Ligustrum vulgare , a world-wide distributed shrub, which inhabits partially shaded areas of the Mediterranean Basin. We measured relevant physiological and biochemical traits, namely: (i) leaf expansion and leaf area expansion rates; (ii) the net CO 2 assimilation rate and the PSII photochemistry; (iii) the concentrations of soluble carbohydrates and photosynthetic pigments; (iv) the activities of superoxide dismutase (SOD), catalase (CAT) and ascorbate peroxidase (APX); (v) the share of assimilated carbon recovered in individual polyphenols; (vi) the leaf oxidative damage. UV-irradiance had a relatively minor impact on most examined traits, as compared with the effect of visible-light irradiance. UV-induced variations in plant growth and net CO 2 assimilation rate were minor. Maximal ( F v / F m ) and actual ( Φ PSII ) efficiencies of PSII photochemistry varied to a greater extent because of visible-light than UV-irradiance, and full-sun leaves had smaller F v / F m and Φ PSII than the partially shaded ones. The conversion state of violaxanthin-cycle pigments was either largely increased by visible-light or unaffected by UV-radiation, as also observed for the activities of antioxidant enzymes (with the exception of SOD). In contrast, UV-radiation greatly enhanced the allocation of carbon to polyphenols, particularly flavonoids, irrespective of visible-light irradiance. Lipid peroxidation and protein oxidation were superior in UV-treated leaves growing under partial shading, whereas leaf oxidative damage was unaffected by UV-radiation in full-sun leaves. We explain the differential UV-induced oxidative damage in partially shaded or full-sun leaves, on the basis of visible-light-induced biochemical adjustments, aimed at avoiding the generation and reducing reactive oxygen forms (ROS). These adjustments included an increase in (1) violaxanthin-cycle pigments, particularly antheraxanthin and zeaxanthin, relative to chlorophyll; (2) antioxidant enzyme activities and flavonoid concentration, which may effectively inhibit the generation and quench ROS once formed.

Mutants with altered leaf morphology are useful as markers for the study of genetic systems and for probing the leaf differentiation process. One such mutant with deficient greening and altered development of the leaf mesophyll appeared in an inbred line of sunflower (Helianthus annuus L.). The objectives of the present study were to determine the inheritance of the mutant leaf trait and its morphological characterisation. The mutation, named mesophyll cell defective1 (mcd1), has pleiotropic effects and it is inherited as a monogenic recessive. The structure and tissue organization of mcd1 leaves are disrupted. In mcd1 leaves, the mesophyll has prominent intercellular spaces, and palisade and spongy tissues are not properly shaped. The mutant palisade cells also appear to be more vacuolated and with a reduced number of chloroplasts than the wild type leaves of equivalent developmental stage. The lamina thickness of mcd1 leaves is greatly variable and in some areas no mesophyll cells are present between the adaxial and abaxial epidermis. The leaf area of the mcd1 mutant is extremely reduced as well as the stem height. A deficient accumulation of photosynthetic pigments characterizes both cotyledons and leaves of the mutant. In mcd1 leaves, chlorophyll (Chl) fluorescence imaging evidences a spatial heterogeneity of leaf photosynthetic performance. Little black points, which correspond to photosystem II (PSII) maximum efficiency (Fv/Fm) values close to zero, characterize the mcd1 leaves. Similarly, the lightadapted quantum efficiency (ΦPSII) values show a homogeneous distribution over wild type leaf lamina, while the damaged areas in mcd1 leaves, represented by yellow zones, are prominent. In conclusion, the loss of function of the MCD1 gene in Helianthus annuus is correlated with a variegated leaf phenotype characterized by a localized destruction of mesophyll morphogenesis and defeat of PSII activity.

Spinach is rich in bioactive constituents such as vitamin C, flavonoids and phenolic acids. In this work, the biochemical modifications occurring during one week of storage at 4 degrees C were evaluated both in intact and in fresh-cut spinach. Results showed that vitamin C concentration is less affected by storage in fresh-cut spinach with respect to intact spinach. MS/MS analysis showed that the main flavonoids are not modified during storage in intact leaves, while some of them increased significantly during storage in the fresh-cut samples. Fresh-cut spinach did not show color alteration even if PPO activity increased significantly during storage. This finding was related to the high ascorbic acid content, which delays the subsequent polymerization events. This finding was confirmed by the unaltered concentration of phenolic compounds in fresh-cut spinach during storage. In conclusion, data about nutritional content and visual performance concurrently suggest that spinach is a suitable species for marketing as a fresh-cut product.

An ozone treatment of 165 nL L −1 for 3 h evoked differential responses in the primary leaves of two bean cultivars of the common bean ( Phaseolus vulgaris L.). While cv ‘Cannellino’ showed visible symptoms of injury within 24 h of exposure, no visible symptoms at all were evident in the cv ‘Top Crop’. In primary leaves of the sensitive cultivar Cannellino, we observed an increase in carbon breakdown (an increase in PFK and Fumarase) and a reduction in CO 2 photoassimilation, linked also to the diminished synthesis of sucrose (a decrease in SPS activity) and to the stimulation of the degradation of this sugar (an increase in SuSy and Invertase activities). A strong stimulation of PEPcase activity indicates both an increased synthesis of OAA and an enhanced replenishment of the tricarboxylic acid cycle. Finally, in Cannellino leaves the activity of NADP-malic enzyme increased indicating a stimulation of enzymes delivering NADPH. The findings of this research suggest that the visible symptoms in Cannellino represent an active response that this cultivar initiates to cope with excess oxidative load. The pattern in Top Crop was different. This cultivar did not show visible symptoms of injury, nor any responsive changes at the physiological and biochemical levels. Oxidative pathways are partially enhanced, e.g., increases in Invertase, PFK and IDH. There were also increases in some enzyme linked to the production of cytosolic NADPH as G6PD, probably caused by the slight increase in activity of the enzymes SKDH and PAL involved in synthesis of phenolic compounds. However, the absence of visible injury in Top Crop leaves is confirmation that, in this cultivar, the need to produce carbon skeleton and NADPH used in detoxification and repair process is lower.

The interactive effects of salinity and potassium deficiency on the growth, mineral elements and photosynthetic performance were investigated in wild (Hordeum maritimum L.) and cultivated barley (Hordeum vulgare L. var. Manel). At 28 d of growth, plants were treated with 3 mM K and 0 mM NaCl (3-0); 3 mM K and 100 mM NaCl (3-100); 0 mM K and 0 mM NaCl (0-0), 0 mM K and 100 mM NaCl (0-100) for 14 d. In both species, biomass production decreased considerably when the two constraints were applied simultaneously. Salinity affected shoots more than roots, whereas for potassium deficiency, the reverse occurred. Generally, potassium uptake was more affected in wild than in cultivated barley and, independent of potassium availability, 100 mM NaCl increased Na+ content in both species, whereas K+ deprivation increased Na(+) content only in H. maritimum shoots (0-0). Potassium-use efficiency (KUE) increased in all treated plants. Potassium deficiency increased the negative effects induced by salt in the photosynthetic process of H. vulgare, and this species seemed to be unable to counteract the negative effects of salinity. H. maritimum showed limitation in CO2 photoassimilation, but this species displayed mechanisms that play a role in avoiding PSII photodamage aimed to dissipate the excess energy.

The interactive effects of root-zone salinity and sunlight on leaf biochemistry, with special emphasis on antioxidant defences, were analysed in Olea europaea L. cv. Allora, during the summer period. Plants were grown outside under 15% (shade plants) or 100% sunlight (sun plants) and supplied with 0 or 125 mM NaCl. The following measurements were performed: (1) the contribution of ions and soluble carbohydrates to osmotic potentials; (2) the photosystem II (PSII) photochemistry and the photosynthetic pigment concentration; (3) the concentration and the tissue-specific distribution of leaf flavonoids; (4) the activity of antioxidant enzymes; and (5) the leaf oxidative damage. The concentrations of Na(+) and Cl(-) were significantly greater in sun than in shade leaves, as also observed for the concentration of the 'antioxidant' sugar-alcohol mannitol. The de-epoxidation state of violaxanthin-cycle pigments increased in response to salinity stress in sun leaves. This finding agrees with a greater maximal PSII photochemistry (F(v)/F(m)) at midday, detected in salt-treated than in control plants, growing in full sunshine. By contrast, salt-treated plants in the shade suffered from midday depression in F(v)/F(m) to a greater degree than that observed in control plants. The high concentration of violaxanthin-cycle pigments in sun leaves suggests that zeaxanthin may protect the chloroplast from photo-oxidative damage, rather than dissipating excess excitation energy via non-photochemical quenching mechanisms. Dihydroxy B-ring-substituted flavonoid glycosides accumulate greatly in the mesophyll, not only in the epidermal cells, in response to high sunlight. The activity of antioxidant enzymes varied little because of sunlight irradiance, but declined sharply in response to high salinity in shade leaves. Interestingly, control and particularly salt-treated plants in the shade underwent greater oxidative damage than their sunny counterparts. These findings, which conform to the evolution of O. europaea in sunny environments, suggest that under partial shading, the antioxidant defence system may be ineffective to counter salt-induced oxidative damage.

CD4+CD25+Foxp3+ regulatory T cells (Treg) are thought to suppress the natural and vaccine-induced immune response against tumor-associated antigens (TAA). Here, we show that Treg accumulate in tumors and tumor-draining lymph nodes of aging transgenic adenocarcinoma of the mouse prostate (TRAMP) male mice, which spontaneously develop prostate cancer. TAA overexpression and disease progression associate also with induction of TAA-specific tolerance. TAA-specific T cells were found in the lymphoid organs of tumor-bearing mice. However, they had lost the ability to release IFN-γ and kill relevant targets. Neither in vivo depletion of Treg by PC61 monoclonal antibody followed by repeated vaccinations with antigen-pulsed dendritic cells nor the combined treatment with 1-methyl-l-tryptophan inhibitor of the enzyme indoleamine 2,3-dyoxigenase, PC61 antibody, and dendritic cell vaccination restored the TAA-specific immune response. Treg did not seem to control the early phases of tolerance induction, as well. Indeed, depletion of Treg, starting at week 6, the age at which TRAMP mice are not yet tolerant, and prolonged up to week 12, did not avoid tolerance induction. A similar accumulation of Treg was found in the lymph nodes draining the site of dendritic cell vaccination both in TRAMP and wild-type animals. Hence, we conclude that Treg accrual is a phenomenon common to the sites of an ongoing immune response, and in TRAMP mice in particular, Treg are dispensable for induction of tumor-specific tolerance. [Cancer Res 2008;68(1):292–300]

Summary Changes in CO2 photo-assimilation and PSII photochemical efficiency in Ramonda serbica leaves during a dehydration–rehydration cycle were examined. The rate of CO2 photo-assimilation was greatly reduced during dehydration, but recovery was complete with rehydration when the relative water content of leaves reached values similar to those of well-hydrated, control leaves. The results showed that the response of R. serbica leaves to severe water stress involves two different mechanisms. In the first, CO2 assimilation is limited by stomata closure that creates an excess proton concentration in the lumen and activates non-photochemical quenching. This plays an important role in the mechanism of photoprotection by dissipation of excitation energy. When dehydration became severe and leaf RWC reached very low values, the electron transport rate (ETR) decreased markedly, while the capacity for regulatory mechanisms such as qNP (non-photochemical quenching) was greatly reduced. For severely dehydrated leaves of R. serbica, it appears that reactive oxygen species (ROS) formation is better prevented by mechanisms that quench chlorophyll triplet formation via lutein.

Summary Eleven peach cultivars (white- and yellow-flesh peaches, nectarines and canning clingstone peaches) were assayed for their antioxidant capacity and their content of some important organic compounds as well as vitamin C, carotenoids and phenols. Antioxidant capacity, determined by FRAP assay, varied between genotypes. Those with the highest value of Ferric Reducing Antioxidant Power (FRAP) were Federica and Springcrest. Antioxidant capacity was correlated to the amount of organic components with significant differences among the cultivars. In some case, antioxidant capacity was related to phenol content, as in yellow–flesh peaches. These results suggest the importance of genotype for determining antioxidant capacity, which, in turn, is related to the organic constituents, such as phenols, vitamin C and carotenoids.

BACKGROUND Poor penetration and uneven distribution of doxorubicin in tumors limits the efficacy of this drug in patients with prostate cancer (PC). Aim of the study was to investigate whether pre-treatment with NGR-TNF, a tumor necrosis factor-α derivative able to target tumor vessels and alter vessel permeability, increases the penetration and the efficacy of doxorubicin in pre-clinical models of PC. METHODS Wild type C57BL/6 mice bearing androgen-independent TRAMP-C1 PC and transgenic adenocarcinoma of the mouse prostate (TRAMP) mice, which spontaneously develop PC and metastasis, were treated with repeated cycles of doxorubicin, administered either alone or following NGR-TNF. Tumor growth and drug uptake by cancer cells was evaluated. RESULTS Doxorubicin as a single agent blocked the growth of TRAMP-C1 cells in vitro but not in vivo. Pre-treatment of mice bearing subcutaneous TRAMP-C1 tumors with NGR-TNF favored doxorubicin penetration into the tumor mass, and in both TRAMP-C1 and TRAMP models significantly delayed tumor growth without increasing drug-related toxicity. CONCLUSIONS Pre-treatment with NGR-TNF significantly expanded the therapeutic index of doxorubicin in mouse models of hormone-dependent and -independent PC. Prostate 68:1105–1115, 2008. © 2008 Wiley-Liss, Inc.

The influence was evaluated of four rootstocks (Ishtara, Mr. S 2/5, GF 677 and Barrier 1) and of harvesting time (early, middle, late) on the quality characteristics and nutritional value (vitamin C, phenols, carotenoids, total antioxidant capacity) of 'Flavorcrest' peach. The better rootstocks were Mr. S 2/5 (low-vigour) and Barrier 1 (high-vigour). In particular, Flavorcrest fruit on Mr. S 2/5 and on Barrier 1 rootstocks had higher antioxidant capacities and also higher phytochemical content, although fruits on Mr. S 2/5 were less firm. Flesh firmness was best for fruits at mid-harvest (H2, 7 July 2006), whereas phytochemical contents were best at late harvest (H3, 13 July 2006), when, for all rootstocks, the best nutritional characteristics were also recorded. Total antioxidant capacity and phytochemical content were determined for the peel and flesh. The results show that removal of peel from peach results in a significant loss of total antioxidant capacity.

Chlorophyll fluorescence has been used routinely to investigate photosynthetic activity in plants subjected to both biotic and abiotic stresses. The aim of this work was to compare the perturbations in photosynthesis induced by ozone and by a pathogen. By using a conventional fluorometer a similar response pattern was observed in inoculated and O 3 -fumigated leaves. The application of chlorophyll fluorescence imaging provided further detailed information on the spatial–temporal heterogeneity of the response of white lupin leaves to fungal pathogen or to ozone fumigation. In particular, 48 h after artificial inoculation with the necrotrophic fungal pathogen Pleiochaeta setosa , the leaves showed a remarkable alteration in PSII operating efficiency (Φ PSII ), which affected the whole surface. Afterwards, the infection site was surrounded by a ring of increased photosynthetic activity. The response of ozonated leaves was quite different. The reduction in Φ PSII was already evident 24 h after fumigation; moreover, a distinct heterogeneity of the fluorescence yield was observed and the major veins displayed a lowered Φ PSII .

Two different genotypes of Lycopersicon esculentum Mill. (cv. Cuor di Bue, O3-sensitive and line 93.1033/1, O3-resistant) were treated with a single dose of ozone (150 mm3 m−3 for 3 h). The PS 2 activity was examined by measurements of chlorophyll a fluorescence on symptomatic and asymptomatic leaves. Symptoms were evident on the 4th leaves from the bottom, in both genotypes, while the 2nd leaves of the line 93.1033/1 were asymptomatic. In these leaves, the net photosynthetic rate (PN) did not change even if the Fv/Fm ratio significantly decreased. A strong reduction in PN, mostly due to the stomatal closure, was observed in Cuor di Bue. The non photochemical quenching coefficient (qNP) and the degree of PS 2 reaction centres closure (1-qP) were higher, while the quantum efficiency of PS 2 photochemistry (ΦPS2) and quantum efficiency of excitation energy capture (Φexc.) were lower in O3 treated leaves of both genotypes. The limitation of photosynthesis was shown also by a decrease in the parameter %P, which diminished compared to controls in both genotypes. The response of the two genotypes for the energy fraction dissipated as thermal energy in the PS 2 antennae (%D) was similar. The fraction of %P remained lower during the recovery in symptomatic leaves of the resistant line as compared to the controls, whereas %X, which represents the amount of light energy that is not utilized in photochemistry or dissipated in the PS 2 antennae, significantly rose in the asymptomatic leaves of this line and in both the leaves of Cuor di Bue. From data obtained we concluded that ozone affected the plants independently on the appearance of visible symptoms of injury because the leaves without visible symptoms of both the genotypes were negatively influenced.

Mutants with a depigmentated phenotype and characterized by Mendelian inheritance can be useful to analyze the role of nuclear genes in chloroplast biogenesis and photosynthesis. In the important oil crop sunflower, we have recently demonstrated that under dim light conditions the xantha1 ( xan1 ) mutant develops true leaves unable to express and retain PSII activity. In order to investigate if this mutation induces an organ specific effect on photosynthetic activity, the light energy utilization of cotyledons (7, 15 and 18 days after sowing) was characterized through gas exchange and chlorophyll a fluorescence. The xan1 cotyledons are unable to fix CO 2 at normal rates. However, rubisco specific activity is not different in the wild-type (wt) and the xan1 seedlings, indicating that biochemical reactions of the photosynthetic process were not altered. The photochemical potential of PSII, measured as the dark-adapted quantum efficiency of PSII ( F v / F m ) was strongly decreased in xan1 cotyledons and is attributable to a significant increase in F 0 values. The light-adapted quantum efficiency ( Φ PSII ) and the efficiency of excitation capture of PSII ( Φ exc. ), were significantly depressed in xan1 cotyledons as compared to the wt. Under light conditions, the most evident result is the strong decrease of Φ PSII , which is linked to a decrease in excitation energy pressure and also to a strong increase in q NP . In summary, with analogy to results previously obtained in leaves, the xan1 cotyledons have a reduced number of photosynthetic units and high sensibility of the PSII reaction centres to photodamage even in low light conditions. We conclude that the loss-of-function of the sunflower XAN1 nuclear gene affects the light energy utilization of chloroplasts in an organ-independent way.

Two differential pathogen-sensitive genotypes of Lycopersicon esculentum plants were treated with a single pulse of ozone (150 nL L−1 for 3 h) and the photosynthetic process and activity of some enzymes of phenylpropanoid metabolism were studied. The sensitive to pathogen variety, Cuor di Bue, showed visible symptoms of injury following the ozone fumigation while the pathogen resistant line 93.1033/1 did not. CO2 fixation ability was compromised in both genotypes but in line 93.1033/1 a complete recovery in photoassimilation of CO2 was observed the day after the end of the fumigation when in Cuor di Bue a further decrease was recorded. No differences between the two tomato lines were found in chlorophyll fluorescence parameters. Shikimate dehydrogenase (SKDH; E.C. 1.1.1.25), l -phenylalanine ammonia-lyase (PAL; E.C. 4.3.1.5) and cinnamyl alcohol dehydrogenase (CAD; E.C. 1.1.1.195) activities in particular were assayed 24 h after the end of the O3 treatment in order to obtain additional information on the biochemical parameters involved in the ozone response. PAL and SKDH increased significantly only in line 93.1033/1 while CAD diminished in both the genotypes. In the resistant line a mechanism attributable to that induced by fungal pathogens was presumable. On the contrary, no clear behavior could be followed for Cuor di Bue.

A series of biochemical parameters, including the concentration of total ascorbic acid (ASA(tot)) and the activities of phenylalanine ammonia lyase (PAL), polyphenol oxidase (PPO), and peroxidases (PODs), was investigated during cold storage (72 h at 4 degrees C in the dark) in fresh-cut (minimally processed) leaves of two lettuce (Lactuca sativa L. var. acephala) cultivars differing in the susceptibility to tissue browning: Green Salade Bowl (GSB), susceptible, and Red Salade Bowl (RSB), resistant. The two cultivars showed differences also at the biochemical level. The content in ASA(tot) increased in RSB, as a consequence of increased DHA concentration; conversely, ASA(tot) diminished in GSB, in which ASA was not detectable after 72 h of storage, thus suggesting a disappearance of ascorbate (both ASA and DHA) into nonactive forms. The antioxidant capacity (as determined by using FRAP analysis) decreased significantly during storage in RSB, while a strong increase was observed in GSB. PAL activity increased soon after processing reaching a maximum by 3 h, then it declined to a relatively constant value in RSB, while in GSB it showed a tendency to decrease in the first few hours from harvest and processing. POD activity, at least for chlorogenic acid, increased significantly during storage only in GSB.

Nuclear mutation xan tha 1 (xan1) arose spontaneously in sunflower and it appeared to have many pleiotropic effects. Homozygous xan1/xan1 seedlings, in field conditions, die after depletion of cotyledonary reserves and can be only recovered by selfing normal heterozygous plants (XAN1/xan1). xan1 mutant showed aberrant development of chloroplasts, deficient pigment content and reduced CO2 assimilation rate. Also, xan1 mutation negatively affected endogenous ABA levels both in non-stressed and dehydrated leaves. Photodestruction of both chlorophyll a and β-carotene in xan1 leaves directly correlated with light intensity. The activation of the xanthophyll cycle under low light (165 PPFD) and the appreciable amounts of zeaxanthin and antheraxanthin retained by xan1 mutant after a 24 h dark period were clear signals of a strong photoinhibition state. The unusual patterns of thylakoid proteins composition detected in seedlings grown under 165 PPFD, by using both denaturing and native electrophoresis analyses, were also indicative of high sensitivity of the thylakoid polypeptide complexes to light stress. Moreover, immunoblot analysis of psbA gene product (D1 protein) indicated that even at 165 PPFD the structure of photosystem (PS) II reaction center was severely damaged. Interestingly, xan1 seedlings grown in dim light (3.5 PPFD) revealed a severely altered chloroplast ultrastructure, despite the lack of photooxidative effects on leaf pigments and the lack of alterations in the electrophoretic profiles of thylakoid proteins. Electron microscopy indicated that XAN1 product is required for normal organization and distribution of thylakoids in the stroma. Chlorophyll a fluorescence analysis pointed to xan1 incompetence to fully express and retain PS II activity, even in the absence of photooxidation.