Effects of UV-B radiation on antioxidant parameters of iron-deficient barley plants

Iron deficiency is a stress frequently experienced by plants, owing to the low solubility of Fe(III) salts in neutral or alcaline soils. Iron is an essential plant nutrient as it is involved in fundamental metabolic processes. Furthermore, it is a constituent of important antioxidant enzymes, which are involved in maintaining the balance of cell redox state. UV-B radiation is an environmental problem which can alter the redox state of plants through the increased production of reactive oxygen species. In order to investigate if iron deficiency influences the antioxidant response of plants to UV-B radiation, barley seedlings, Hordeum vulgare L. cv. Express, were exposed to UV-B radiation while growing in nutrient solutions with or without iron. After eight days of growth, plants were harvested and analysed. Results show that, during the 8 days of the experimental period, in neither of the two nutritional conditions considered does UV-B exposure reduce shoot weight or induce evident alterations of thylakoid membranes in respect to controls. However, different responses to UV-B radiation between iron-deficient and iron-sufficient plants were observed at the level of parameters related to oxidative stress. In fact, in iron-sufficient plants the contents of photosynthetic pigments and ascorbate, and the enzyme activities of ascorbate peroxidase (EC 1.11.1.11) and catalase (EC 1.11.1.6) were not affected by UV-B radiation. Conversely, in iron-deficient plants the contents of ascorbate and zeaxanthin and the activity of ascorbate peroxidase increased under UV-B exposure, whereas catalase activity decreased. Furthermore, UV-B radiation induced an increase of hydrogen peroxide content which was higher in iron-deprived plants than in iron-sufficient ones. This may indicate that plants growing in an environment enriched in UV-B radiation may develop a high level of oxidative stress when iron supply is limited.     

Obestatin regulates cardiovascular function and promotes cardioprotection through the nitric oxide pathway

Patients with ischaemic heart disease or chronic heart failure show altered levels of obestatin, suggesting a role for this peptide in human heart function. We have previously demonstrated that GH secretagogues and the ghrelin gene‐derived peptides, including obestatin, exert cardiovascular effects by modulating cardiac inotropism and vascular tone, and reducing cell death and contractile dysfunction in hearts subjected to ischaemia/reperfusion (I/R), through the Akt/nitric oxide (NO) pathway. However, the mechanisms underlying the cardiac actions of obestatin remain largely unknown. Thus, we suggested that obestatin‐induced activation of PI3K/Akt/NO and PKG signalling is implicated in protection of the myocardium when challenged by adrenergic, endothelinergic or I/R stress. We show that obestatin exerts an inhibitory tone on the performance of rat papillary muscle in both basal conditions and under β‐adrenergic overstimulation, through endothelial‐dependent NO/cGMP/PKG signalling. This pathway was also involved in the vasodilator effect of the peptide, used both alone and under stress induced by endothelin‐1. Moreover, when infused during early reperfusion, obestatin reduced infarct size in isolated I/R rat hearts, through an NO/PKG pathway, comprising ROS/PKC signalling, and converging on mitochondrial ATP‐sensitive potassium [mitoK(ATP)] channels. Overall, our results suggest that obestatin regulates cardiovascular function in stress conditions and induces cardioprotection by mechanisms dependent on activation of an NO/soluble guanylate cyclase (sGC)/PKG pathway. In fact, obestatin counteracts exaggerated β‐adrenergic and endothelin‐1 activity, relevant factors in heart failure, suggesting multiple positive effects of the peptide, including the lowering of cardiac afterload, thus representing a potential candidate in pharmacological post‐conditioning.     

Perfusion of isolated rat kidney with Mesenchymal Stromal Cells/Extracellular Vesicles prevents ischaemic injury

Kidney donation after circulatory death (DCD) is a less than ideal option to meet organ shortages. Hypothermic machine perfusion (HMP) with Belzer solution (BS) improves the viability of DCD kidneys, although the graft clinical course remains critical. Mesenchymal stromal cells (MSC) promote tissue repair by releasing extracellular vesicles (EV). We evaluated whether delivering MSC‐/MSC‐derived EV during HMP protects rat DCD kidneys from ischaemic injury and investigated the underlying pathogenic mechanisms. Warm ischaemic isolated kidneys were cold‐perfused (4 hrs) with BS, BS supplemented with MSC or EV. Renal damage was evaluated by histology and renal gene expression by microarray analysis, RT‐PCR. Malondialdehyde, lactate, LDH, glucose and pyruvate were measured in the effluent fluid. MSC‐/EV‐treated kidneys showed significantly less global ischaemic damage. In the MSC/EV groups, there was up‐regulation of three genes encoding enzymes known to improve cell energy metabolism and three genes encoding proteins involved in ion membrane transport. In the effluent fluid, lactate, LDH, MDA and glucose were significantly lower and pyruvate higher in MSC/EV kidneys as compared with BS, suggesting the larger use of energy substrates by MSC/EV kidneys. The addition of MSC/EV to BS during HMP protects the kidney from ischaemic injury by preserving the enzymatic machinery essential for cell viability and protects the kidney from reperfusion damage.     

LPS,Oleuropein and Blueberry extracts affect the survival,morphology and Phosphoinositide signalling in stimulated human endothelial cells

Endothelial cells (EC) act as leading actors in angiogenesis. Understanding the complex network of signal transduction pathways which regulate angiogenesis might offer insights in the regulation of normal and pathological events, including tumours, vascular, inflammatory and immune diseases. The effects of olive oil and of Blueberry extracts upon the phosphoinositide (PI)-specific phospholipase C (PLC) enzymes were evaluated both in quiescent and inflammatory stimulated human umbilical vein EC (HUVEC) using molecular biology (multiliquid bioanalysis) and immunofluorescence techniques. Oleuropein significantly increased the number of surviving HUVEC compared to untreated controls, suggesting that it favours the survival and proliferation of EC. Our results suggest that Oleuropein might be useful to induce EC proliferation, an important event during angiogenesis, with special regard to wound healing. Blueberry extracts increased the number of surviving HUVEC, although the comparison to untreated controls did not result statistically significant. Lipopolysaccharide (LPS) administration significantly reduced the number of live HUVEC. LPS can also modify the expression of selected PLC genes. Adding Blueberry extracts to LPS treated HUVEC cultures did not significantly modify the variations of PLC expression induced by LPS. Oleuropein increased or reduced the expression of PLC genes, and statistically significant results were identified for selected PLC isoforms. Oleuropein also modified the effects of LPS upon PLC genes’ expression. Thus, our results corroborate the hypothesis that Oleuropein owns anti-inflammatory activity. The intracellular localization of PLC enzymes was modified by the different treatments we used. Podosome-like structures were observed in differently LPS treated HUVEC.     

Zymographic analysis of circulating and tissue forms of colon carcinoma gelatinase A (MMP-2) and B (MMP-9) separated by mono- and two-dimensional electrophoresis.

Gelatinase A (MMP-2) and gelatinase B (MMP-9) play a key role in the proteolytic cascade leading to ECM degradation during invasion and metastasis. The enzyme activity is regulated both at the intra- and extra-cellular level. Extracellular regulation is achieved mainly through the balance between proenzyme activation and inhibition, which appears to be altered in cancer patients. One of the mechanisms of MMP inhibition is the binding of the enzymes to appropriate tissue inhibitors (TIMP). In the recent literature, it has been suggested that MMP-2 and/or MMP-9 are indeed over-produced in many carcinomas, while the identity of the various enzymatic forms (latent, activated and enzyme/inhibitor complexes) remains to be elucidated. In this study we have analyzed the circulating forms of MMP-9 and MMP-2 in serum samples of patients with colon carcinoma, as well as the enzymatic activities present in tissue extracts from surgical fragments (primary tumor and its paired healthy tissue). Proteins were separated by means of mono-dimensional or bidimensional electrophoresis, and the enzymes detected by gelatin zymography and immunological assays. The results of densitometric analyses demonstrate that proMMP-9, but not proMMP-2, is significantly higher in the oncologic sera vs. the normal sera. In addition, several oligomeric circulating and tissue forms of MMP-9 are preferentially found in the oncologic samples, both in mono- and second-dimension zymograms. The activated forms of MMP-2 and MMP-9 are uniquely present in the primary tumor extracts, thus confirming the involvement of the tissue microenvironment in gelatinase activation and function.     

Molecular Investigation and Phylogeny of Anaplasma spp. in Mediterranean Ruminants Reveal the Presence of Neutrophil-Tropic Strains Closely Related to A. platys

Few data are available on the prevalence and molecular typing of species belonging to the genus Anaplasma in Mediterranean ruminants. In this study, PCR analysis and sequencing of both 16S rRNA and groEL genes were combined to investigate the presence, prevalence, and molecular traits of Anaplasma spp. in ruminants sampled on the Island of Sardinia, chosen as a subtropical representative area. The results demonstrate a high prevalence of Anaplasma spp. in ruminants, with animals infected by at least four of six Anaplasma species (Anaplasma marginale, A. bovis, A. ovis, and A. phagocytophilum). Moreover, ruminants host a number of neutrophil-tropic strains genetically closely related to the canine pathogen A. platys. The high Anaplasma spp. prevalence and the identification of as-yet-unclassified neutrophil-tropic strains raise concerns about the specificity of serological tests routinely used in ruminants and provide additional background for reconstructing the evolutionary history of species genetically related to A. phagocytophilum.     

Responses of the Antarctic microalga Koliella antarctica (Trebouxiophyceae,Chlorophyta) to cadmium contamination

Ultrastructural and physiological effects of exposure to 1 ppm and 5 ppm of cadmium (Cd) on cultured cells of Koliella antarctica, a green microalga from Antarctica, were investigated. The amount of Cd in the alga rose with the increase of the metal concentration in the growth medium and most Cd remained outside the cells, bound to the components of the cell walls. The increase of Cd in the microalga was concomitant with the decrease of other elements, mainly calcium (Ca). Exposure to 1 ppm Cd slowed culture growth by inhibiting cell division and also caused the development of some misshapen cells with chloroplast showing disordered thylakoids. However, this concentration did not substantially affect the chlorophyll (Chl) content or photosystem (PS) activity. At 5 ppm, Cd cell growth suddenly stopped and some cells lysed. After a week of Cd contamination, the cells were enlarged and severely damaged. The chloroplasts showed great ultrastructural alterations and a reduced Chl content. Cd exposure negatively affected PSII, whose activity was almost completely lost after four days.