Molecular characterization of Pseudomonas fluorescens isolates involved in the Italian "blue mozzarella" event.

Between June and September 2010, widespread Italian consumer reports of unusual blue spoilage on fresh dairy products were publicized, resulting in the so-called blue mozzarella event. An inordinately high number of samples from mozzarella and whey cheese products of Italian and German production subsequently tested positive for Pseudomonas fluorescens. The aim of this study was to verify whether a selected P. fluorescens strain was responsible for this apparently unusual event. Molecular characterization of 181 isolated P. fluorescens strains was conducted using a newly optimized pulsed-field gel electrophoresis protocol. Although a high number of pulsotypes was found (132), only four pulsotypes were associated with more than one production plant, and only one German isolate had the same pulsotype as was detected in two Italian plants. This is the only evidence of possible cross-contamination among cheeses from the two countries. The overall results did not support the spread of contamination from German to Italian plants or the presence of one environmental strain that spread in both countries.

TLR2: a crossroads between infections and autoimmunity?

Environment has both pathogenic and protective roles in the determination of autoimmune disease development, possibly through infectious agents. TLR2 has the capability to recognize the widest range of PAMPs, and it is important for the recognition of mycobacteria and gram-positive bacteria. Here we review recent information showing that TLR2 ligands, its signaling machinery and the effects of its engagement on T cell polarization and differentiation, all play a decisive role in experimental models of autoimmunity. Thus, we propose that engagement of TLR2 is an important crossroad between encounter with bacteria and development of self-reactive diseases.

The challenge of education for the veterinary inspector.

The aim of this review is to provide an insight into the current state of, and future changes in, veterinary education. At present, Veterinary Inspectors need to have the appropriate education, relevant experience and the ability to carry out their duties in the context of inspections. They should respond to the changes in legislation related to food and to the official control systems and must take into account the Community rules. In effect, there is still a wide diversity among schools in Italy and in Europe, showing that there is an urgent need for harmonization of the veterinary education, in order to have a common basis of knowledge. Moreover, it is important to maintain and update all knowledge by means of the so-called lifelong learning. Along the educational route of the veterinary inspector, many gaps still exist: the entire system of learning should be revised, with the aim of having a well-integrated education system in cooperation with all the figures involved. In this way Veterinary Inspectors will be able to face the new issues posed by society and the globalization of food consumption and will be able to maintain their position in the assurance of food safety.

Pro-metastatic signaling by c-Met through RAC-1 and reactive oxygen species (ROS).

Overexpression of the c-Met/hepatocyte growth factor receptor(HGF-R) proto-oncogene and abnormal generation of intracellular oxygen species (reactive oxygen species (ROS)) have been linked, by independent lines of evidence, to cell transformation and to malignant growth. By comparing two subpopulations of the B16 mouse melanoma (B16-F0 and B16-F10) endowed with different lung metastasis capacities (low and high, respectively) we found that both the expression/phosphorylation of c-Met and the steady-state levels of ROS positively correlated with metastatic growth. shRNA-mediated downregulation of c-Met in F10 cells led to a parallel decrease in the generation of oxygen species and in metastatic capacity, suggesting that oxidants may mediate the pro-metastatic activity of the HGF receptor. c-Met activation by a ligand elicits the formation of oxidant species through the oxidase-coupled small GTPase Rac-1, a relevant downstream target of the HGF-R. Moreover, cell treatment with the catalytic ROS scavengers EUK-134 and EUK-189 attenuates Met signaling to ERKs and inhibits the anchorage-independent growth of F10 cells, consistent with a critical role for oxygen species in HGF signaling and in aggressive cell behavior. Finally, genetic manipulation of the Rac-ROS cascade at different levels demonstrated its crucial role in the pro-metastatic activity of c-Met in vivo. Thus, we have outlined a novel cascade triggered by c-Met and mediated by ROS, linked to metastasis and potentially targetable by new antimetastatic, redox-based therapies.

Protective role of MnSOD and redox regulation of neuronal cell survival.

Reactive oxygen species (ROS) play a central role in neuronal pathophysiology and in neurodegenerative disorders. However, recent evidence indicates that these molecules also operate as signaling intermediates in a variety of physiological settings, including cell protection from apoptosis. Data presented here strongly support such a dual role for oxidants in neuronal cell homeostasis. In rat pheocromocytoma cells, cell rescue by the nerve growth factor (NGF) is accompanied by a transient burst of ROS generated in the cytosol by a GTPase-dependent mechanism. Within the NGF signaling cascade, ROS lie upstream and are necessary for activation/phosphorylation of AKT/PKB and of the antiapoptotic transcription factor cAMP-responsive element-binding protein (CREB). Conversely, an increase in mitochondrial oxygen species heralds apoptosis of serum-deprived cells, and these events can be prevented by cell exposure to NGF or by treatment with the mitochondrially targeted antioxidant MitoQ. Importantly, NGF-mediated decrease of mitochondrial ROS is dependent on the transcriptional up-regulation of the manganese superoxide dismutase (MnSOD) by active CREB. These observations therefore outline a circuitry whereby cytosolic redox signaling promotes neuronal cell survival by increasing the mitochondrial antioxidant defenses.

The level of manganese superoxide dismutase content is an independent prognostic factor for glioblastoma. Biological mechanisms and clinical implications.

We address the issue of the role of manganese superoxide dismutase in tumorigenesis by studying a relatively homogeneous group of tumours for the correlation between amount of this anti-oxidant enzyme and prognosis. The clinical outcome of 30 patients affected by glioblastomas whose manganese superoxide dismutase content had been established at the time of first diagnosis is compared. When the survival of patients is stratified according to manganese superoxide dismutase level in the tumour, a link of these levels and prognosis can be observed. Patients with high levels of manganese superoxide dismutase show a median survival time of 6.11 months, while patients whose tumours display a low amount of MnSOD have a median survival time of 12.17 months. To assess the upstream mechanisms that sustain the increase in manganese superoxide dismutase content in brain neuroepithelial tumours, we also studied the expression of p53 in a series of 17 astrocytomas of various grading. In all tested astrocytomas, high manganese superoxide dismutase content is associated with cytoplasmic accumulation of p53. Thus glioblastomas can be divided into two distinct groups on the basis of their content of manganese superoxide dismutase, having 'better' or 'worse' prognosis, respectively. The use of this protein as a marker may help to define therapeutic strategies in the clinical management of glioblastoma.

Cell compartmentalization in redox signaling.

From a growing body of evidence on the role of Reactive Oxygen Species as intracellular signaling molecules, the concept starts to emerge that cell responses to redox changes are function of the intracellular site where oxidants are produced and/or meet their molecular targets. In particular, a major distinction between oxidative events in the cytosolic versus the mitochondrial compartment appears to exist in terms of physiological stimuli, signaling mechanisms and functional consequences. Experimental data supporting this view are reviewed here, and the potential implications of this new perspective in redox signaling are discussed.

Deregulated manganese superoxide dismutase expression and resistance to oxidative injury in p53-deficient cells.

Loss of function of the tumor suppressor protein p53 represents a very frequent event in human carcinogenesis, but the molecular mechanisms linking impaired p53 activity to increased cell malignancy are still incompletely understood. p53 is normally involved in both cell cycle control and the induction of cell death and is involved in the latter mainly through the transcriptional regulation of pro- and antiapoptotic proteins. Reactive oxygen species are known to be powerful inducers of p53 activity; moreover, they play a role in the execution of p53-dependent apoptosis. Here we show that transformed mouse fibroblasts lacking p53 are significantly more resistant than wild-type (wt) controls to the cytotoxic effect of a number of pro-oxidant treatments. Interestingly, these cells also exhibit deregulated expression of the antioxidant enzyme manganese superoxide dismutase (MnSOD), a protein known to protect cancer cells from the oxidative injury inflicted by antitumoral cytokines and anticancer drugs. MnSOD activity was also increased in liver tissue from p53-deficient mice in comparison with wt tissue. Transient transfection of wt p53 in HeLa cells led to a significant reduction in steady-state MnSOD mRNA levels and enzymatic activity, confirming that the expression of this antioxidant enzyme is negatively regulated by p53. Forced expression of MnSOD rendered HeLa cells resistant to p53-dependent cytotoxic treatments and, in cotransfection experiments, counteracted the growth-inhibitory effect of p53. Taken together, these data identify MnSOD as a potential target for tumor suppressor protein p53 and underscore the relevance of MnSOD modulation in the context of normal p53 functions because it is consistent with many reports of abnormally increased MnSOD expression in human cancers.

A redox signaling mechanism for density-dependent inhibition of cell growth.

Reactive oxygen species (ROS) have recently drawn significant attention as putative mitogenic mediators downstream of activated growth factor receptors and oncogenic Ras; however, the possibility that a redox-related mechanism also operates in the negative control of cell proliferation by inhibitory signals has not been investigated thus far. Here we show that the arrest of growth induced by cell confluence ("contact inhibition") is due, at least in part, to a decrease in the steady-state levels of intracellular ROS and the consequent impairment of mitogenic redox signaling. In confluent fibroblast cultures, the decrease in the concentration of oxygen species was associated with diminished activity of the small GTPase Rac-1, a signal transducer directly involved in the ligand-dependent generation of oxygen-derived molecules, and was effectively mimicked by exposure of sparse cultures to dithiothreitol (DTT) and inhibitors of enzymes (phospholipase A2 and lipoxygenase) acting in the arachidonic acid cascade downstream of growth factor receptors and Rac-1. Sparse fibroblasts treated with nontoxic amounts of DTT underwent growth arrest, whereas a low concentration of hydrogen peroxide significantly increased thymidine incorporation in confluent cultures, demonstrating a causal link between redox changes and growth control by cell density. Removal of oxygen species from sparse cultures was accompanied by a drastic decrease of protein tyrosine phosphorylation after epidermal growth factor stimulation, which, at a biochemical level, reproduced the signaling hallmarks of contact inhibition. Moreover, the cytosolic tyrosine phosphatase SHP-2 was identified as a putative target for redox signaling by cell density because the enzyme itself and the associated substrates appear markedly dephosphorylated in both confluent and reductant-treated cells after exposure to epidermal growth factor, and SHP-2 enzymatic activity is strongly activated by DTT in vitro. Taken together, these data support a model in which impaired generation of ROS and increased protein tyrosine phosphatase activity impede mitogenic signaling in contact-inhibited cells.

Redox regulation of lymphocyte signaling.

Compelling evidence exists that reactive oxygen species can deliver intracellular signals in mammalian cells, and elicit a broad array of physiological responses according to the cell type, the oxidative burden and the cellular compartment where radicals are generated. When applied to immune cells, these concepts gain a particular relevance, in relation to the plasticity of immune functions and the biological complexity of lymphocyte response to antigens. Here we review some recent and somehow conflicting observations on the involvement of oxygen radicals and redox balance in lymphocyte activation, and propose models for how radical species could contribute to normal and pathological immunity.

Endogenous oxygen radicals modulate protein tyrosine phosphorylation and JNK-1 activation in lectin-stimulated thymocytes.

Molecular events mediating the T-lymphocyte response to lectins are still incompletely understood, although much evidence suggests that both the mitogenic and the death-promoting effects of these agents involve the biochemical cascade initiated by the CD3/T-cell antigen receptor (TCR) complex. Reactive oxygen species (ROS) and in particular H(2)O(2) have been shown to have a role in cell response to cytokines and growth factors. Here we report that the proliferation of mouse thymocytes in response to the mitogenic lectin concanavalin A (ConA) is strongly and selectively inhibited by the intracellular ROS scavenger N-acetylcysteine (NAC) and by diphenyleneiodonium (DPI), a potent inhibitor of NADPH-dependent membrane oxidases activated by surface receptors. A rapid 'burst' of intracellular oxygen radicals was observed in mouse thymocytes stimulated by ConA, with kinetics that paralleled the appearance of tyrosine-phosphorylated proteins. This burst was abrogated by the pretreatment of cells with NAC or DPI. Only a modest increase in intracellular oxygen species was found in thymocytes stimulated by strong cross-linking of TCR together with CD4 or CD28. Pharmacological interference with ROS production in ConA-stimulated thymocytes resulted in a decreased tyrosine phosphorylation of multiple protein species, including a 38 kDa band able to recruit the adapter protein Grb2 and corresponding to the recently identified transducer LAT (linker for activation of T-cells), a molecule involved in linking activated TCR to the production of interleukin 2 and the proliferation of T-cells. Furthermore, ROS inhibition markedly attenuated the activation of stress-activated protein kinase/JNK-1 (c-Jun N-terminal kinase 1) in response to lectins. Taken together, these results identify ROS as important modulators of the signalling cascade initiated by mitogenic lectins in thymocytes and, by extension, as a novel class of mediators downstream of antigen receptors.

Increased growth capacity of cervical-carcinoma cells over-expressing manganous superoxide dismutase.

Increases in the expression of manganese-dependent superoxide dismutase (MnSOD) have been detected in several classes of human and experimental tumors and appear to correlate with poorer prognosis in human neuro-epithelial, ovarian and cervical tumors. To delineate the relevance of MnSOD expression to tumor-cell growth and survival, a human MnSOD cDNA was over-expressed in the HeLa cervical-carcinoma cell line. MnSOD over-expression had marginal effects on the growth of HeLa cells in standard medium, but markedly protected the cells from growth suppression and cell death in conditions of serum deprivation. Serum starvation did not affect expression of endogenous MnSOD in wild-type HeLa cells, but was associated with increases in cell death and in the generation of intracellular oxygen radicals. By contrast, in HT29 colon-carcinoma cells, which are relatively resistant to growth-factor withdrawal, serum deprivation was associated with increases in MnSOD expression and activity. Together these observations suggest that MnSOD provides a mechanism for counteracting the intracellular oxidative processes that impair cell growth and viability in the context of growth-factor withdrawal and, in this context, may promote tumor-cell survival in vivo in conditions normally unfavorable to cell growth.

Oxidative stress and overexpression of manganese superoxide dismutase in patients with Alzheimer's disease.

Substantial evidence supports the hypothesis that oxygen free radicals are involved in various neurodegenerative disorders. To assess the presence of oxidative stress in Alzheimer's disease (AD) we examined the activity of the enzyme copper-zinc superoxide dismutase (CuZnSOD) in red blood cells, the levels of the mitochondrial inducible enzyme manganese superoxide dismutase (MnSOD) mRNA in lymphocytes, and the total radical-trapping antioxidant capacity (TRAP) in plasma of AD patients and in a group of age-matched non-demented controls. We found that CuZnSOD activity (P < 0.01 vs. controls) was significantly increased as well as the MnSOD mRNA levels while the total antioxidant status (P < 0.001 vs. controls) was decreased in AD patients. These findings support the role of oxidative alterations in the pathogenetic mechanism underlying AD neurodegeneration.

Iron modulation of LPS-induced manganese superoxide dismutase gene expression in rat tissues.

Mitochondrial superoxide dismutase (MnSOD) is usually diminished in cancer cells. We observed that in vivo treatment with LPS produces a strong increase of MnSOD mRNA levels and a weak induction of an inactive protein in rat hepatocarcinomas. In normal liver iron deficiency, obtained with desferrioxamine administration, produces a decrease in the MnSOD induction by LPS, indicating that such induction could depend on tissue iron content. However, no change in MnSOD mRNA has been observed in iron-overloaded tumor tissue. Thus, iron is possibly involved in the transcriptional regulation of the protein, in combination with some other unknown factor that appears to be deficient in tumor cells.

NF kappa B-independent transcriptional induction of the human manganous superoxide dismutase gene.

Numerous conditions induce expression of manganese-containing superoxide dismutase (MnSOD) in mammalian cells. The reported inducers of MnSOD are all agents that activate two transcription factors, AP-1 and NF kappa B, but several reports have suggested that MnSOD induction relies solely on NF kappa B. We investigated the contribution of the individual transcription factors by using antioxidants and metal chelators to modulate MnSOD transcriptional activation in response to phorbol esters or hydrogen peroxide. The results indicate substantial transcriptional induction of the MnSOD gene independent of NF kappa B. The metal chelator and antioxidant pyrrolidine dithiocarbamate (PDTC) at 60 or 100 microM induced the MnSOD transcript in HeLa cells while diminishing expression of the NF kappa B-responsive transcript I kappa B-alpha. Induction of the MnSOD mRNA by phorbol-12-myristate-13-acetate (PMA) was only slightly diminished in the presence of PDTC, which in contrast virtually eliminated induction of the NF kappa B-dependent transcript I kappa B-alpha by PMA. MnSOD RNA induction by H2O2 was only approximately 1.5-fold, compared to a ca. 3-fold activation of I kappa B-alpha expression. Two other antioxidants, N-acetyl-L-cysteine and butylated hydroxyanisole, failed to block induction of the MnSOD transcript by PMA, which is consistent with a role for AP-1. In vitro DNA binding studies confirmed strong AP-1 activation under conditions where NF kappa B is blocked but the MnSOD transcript is strongly induced (e.g., PMA treatment in the presence of PDTC).

Diethyldithiocarbamate treatment up regulates manganese superoxide dismutase gene expression in rat liver.

In vivo experiments demonstrate that rat liver manganese-containing superoxide dismutase (MnSOD) is up-regulated at the transcriptional level following the inactivation of copper-zinc superoxide dismutase (CuZnSOD). CuZnSOD activity was inhibited by the administration of the copper chelating agent diethyldithiocarbamate (DDC). This CuZnSOD inactivation is likely associated with an intracellular oxidative stress. Indeed the antioxidant N-acetyl-cysteine (NAC) completely prevents the MnSOD mRNA up-regulation observed after DDC administration. Evidence is also provided that an approximately 50% diminution of the total iron content in the tissue, which follows the in vivo administration of the iron chelator desferrioxamine (DESF), reduces the amount of MnSOD induction achieved by DDC treatment. Both NAC and DESF significantly down-regulate MnSOD gene expression also in normal untreated rat liver. While the observed inhibitory effect of NAC in MnSOD mRNA up-regulation can be ascribed mainly to its antioxidant property, iron chelation could act with an antioxidant effect and/or affecting some iron-dependent factor(s) possibly involved in MnSOD gene regulation. It is proposed that this metal could have a role among factors that sense and/or trigger transcription of the MnSOD gene.

Ethanol treatment up-regulates the expression of mitochondrial manganese superoxide dismutase in rat liver.

On the basis of the well-known effect of ethanol poisoning on the prooxidant/antioxidant balance of human and rodent liver we tested the response of the mitochondrial manganese-containing superoxide dismutase (MnSOD) in the liver of rats following an acute ethanol load or chronically intoxicated with an alcohol-supplemented solid diet for three weeks. In both conditions the enzyme activity and messenger RNA were monitored. In the acutely treated animals MnSOD was induced (post-)translationally already at 3 hours after ethanol injection, reached the maximum level (about 50% increment) at 9 hours and decreased thereafter. Chronic ethanol feeding caused an up-regulation of the enzyme at the mRNA level, with a good correlation between the transcript and the enzyme activity during the first two weeks of treatment. After 20 days the mRNA level dropped to normal, whereas the activity still remained high. Chronic alcohol intake also led to a significant decrease in the content of vitamin E in the liver mitochondrial and microsomal fractions, suggesting the occurrence of an enhanced lipid peroxidation, consequent to the ethanol-induced oxidative stress. The response of MnSOD appears to be a protective mechanism that the genetic machinery builds up to partially overcome such a condition.

Defective gene expression of MnSOD in cancer cells.

Reactive oxygen species (ROS) have been frequently implicated in the initiation and promotion phases of carcinogenesis. Antioxidant enzymes, which can antagonize this process, are lowered in a number of malignancies. The enzyme most commonly decreased is the mitochondrial Mn-containing superoxide dismutase (MnSOD) encoded by a nuclear gene mapped on the band 6q21, a region frequently deleted in several human tumours. The close association of del(6q) with diminution of MnSOD has led to suggest that MnSOD might be a new type of tumour-suppressor gene. This hypothesis is also sustained by the finding that transfection of MnSOD cDNA into human melanoma cell lines suppress the malignant phenotype. There are, however, conflicting observations that tend to ascribe the deficiency of the MnSOD activity more to a defect in the expression of the gene than to its deletion. In many transformed cell lines, including some with marked del(6q), there is no change in the dosage of the MnSOD gene and the enzyme is highly inducible by various pro-oxidant agents. Transition metals (Mn, Fe) have been found to be highly deficient in human and rodent tumours. Owing to the second messenger function of ROS in activating transcription factors (NF-kB, AP-1) and to the ability of Mn to facilitate the dismutation of O2- to H2O2 and of Fe to participate in the Fenton reaction, we propose that in the early stage of carcinogenesis an impairment of the signal transduction machinery, related to the metal deficiency, might limit the binding to DNA of transcription factors and cause the defect in the MnSOD gene expression.

Transcriptional regulation of MnSOD by manganese in the liver of manganese-deficient mice and during rat development.

Evidence is reported that liver manganese deficiency, whether artificially produced by the administration of a Mn-deficient diet, or physiologically occurring in the neonatal life, in mice and rats respectively, causes the down-regulation of the manganese-containing superoxide dismutase at (pre)-transcriptional level. These observations, in addition to previous data concerning Mn-deficiency and the low level of expression of MnSOD in Morris hepatomas, strongly support the role played by the metal ion in the control of the MnSOD by a mechanism of gene activation. While the molecular events taking place in such regulation are not yet identified, the involvement of reactive oxygen species (ROS) as second messengers in the activation of specific transcription factors is suggested.

Manganese deficiency and transcriptional regulation of mitochondrial superoxide dismutase in hepatomas.

The presumed involvement of the transition metals manganese and copper in the regulation of the expression of the Mn- and CuZn-containing superoxide dismutase genes has been investigated in normal and neoplastic tissues of the rat. Two hepatomas of the Morris line have been employed, the slow growing, highly differentiated 9618A and the fast growing, poorly differentiated 3924A. The data obtained indicate a control at the pretranslational level of the Mn-containing enzyme, presumably exerted by the manganese ion. The CuZn-containing superoxide dismutase is also regulated pretranslationally in the normal tissues examined and in the hepatoma 3924A. However, there is no indication for the involvement of the copper ion, which in the liver is mostly located in the cytosol bound to CuZnSOD, in such regulation. The possible role of a reduced redox state, concomitant to the manganese deficiency in hepatoma tissues, in the down regulation of Mn-containing superoxide dismutase is discussed.