Azione antiossidante del sulforafane: analisi in cellule cardiache in coltura ed in un modello animale di esercizio fisico

Oxidative stress is considered to be of major relevance for a variety of pathological processes. Thus, it is valuable to identify compounds, which might act as antioxidants, i.e. compounds that antagonize the deleterious action of reactive oxygen species (ROS) on biomolecules. The mode of action of these compounds could be either to scavenge ROS directly or to trigger protective mechanisms inside the cell, thereby resulting in improved defense against ROS. Sulforaphane (SF) (1-isothiocyanato-(4R)-(methylsulfinyl)butane) is a naturally occurring cancer chemopreventive agent found as a precursor glucosinolate in Cruciferous vegetables like broccoli. Although SF is not a direct-acting antioxidant, there is substantial evidence that SF acts indirectly to increase the antioxidant capacity of animal cells and their abilities to cope with oxidative stress. Induction of phase 2 enzymes is one means by which SF enhances the cellular antioxidant capacity. Enzymes induced by SF include Glutathione S-transferases (GST) and NAD[P]H:quinone oxidoreductase (NQO1) which can function as protectors against oxidative stress. To protect themselves from oxidative stress, cells are equipped with reducing buffer systems including the GSH and thioredoxin (Trx) reductase. GSH is an important tripeptide thiol which in addition to being the substrate for GSTs maintains the cellular oxidation– reduction balance and protects cells against free radical species. Aim of the first part of this thesis was to investigate the ability of SF to induce the expression and the activity of different phase 2 and antioxidant enzymes (such as GST, GR, GPx, NQO1, TR, SOD, CAT) in an in vitro model of rat cardiomyocytes, and also to define if SF treatment supprts cells in counteracting oxidative stress induced by H2O2 It is well known that acute exhaustive exercise causes significant reactive oxygen species generation that results in oxidative stress, which can induce negative effects on health and well being. In fact, increased oxidative stress and biomarkers (e.g., protein carbonyls, MDA, and 8- hydroxyguanosine) as well as muscle damage biomarkers (e.g. plasmatic Creatine cinase and Lactate dehydrogenase) have been observed after supramaximal sprint exercises, exhaustive longdistance cycling or running as well as resistance-type exercises, both in trained and untrained humans. Markers of oxidative stress also increase in rodents following exhaustive exercise. Moreover, antioxidant enzyme activities and expressions of antioxidant enzymes are known to increase in response to exhaustive exercise in both animal and human tissues. Aim of this project was to evaluate the effect of SF supplementation in counteracting oxidative stress induced by physical activity through its ability to induce phase 2, and antioxidant enzymes in rat muscle. The results show that SF is a nutraceutical compound able to induce the activity of different phase 2 and antioxidant enzymes in both cardiac muscle and skeletal muscle. Thanks to its actions SF is becoming a promising molecule able to prevent cardiovascular damages induced by oxidative stress and muscle damages induced by acute exhaustive exercise.

Variation in peach fruit susceptibility to Monilinia laxa and gene expression

Brown rot caused by Monilinia laxa and Monilinia fructigena is considered one of the most important diseases affecting Prunus species. Although some losses can result from the rotten fruits in the orchard, most of the damage is caused to fruits during the post-harvest phase. Several studies reported that brown rot incidence during fruit development highly varies; it was found that at a period corresponding to the the pit hardening stage, fruit susceptibility drastically decreases, to be quickly restored afterwards. However the molecular basis of this phenomenon is still not well understood. Furthermore, no difference in the rot incidence was found between wound and un-wound fruits, suggesting that resistance associated more to a specifc biochemical response of the fruit, rather than to a higher mechanical resistance. So far, the interaction Monilinia-peach was analyzed through chemical approaches. In this study, a bio-molecular approach was undertaken in order to reveal alteration in gene expression associated to the variation of susceptibility. In this thesis three different methods for gene expression analysis were used to analyze the alterations in gene expression occurring in peach fruits during the pit hardening stage, in a period encompassing the temporary change in Monilinia susceptibility: real time PCR, microarray and cDNA AFLP techniques. In 2005, peach fruits (cv.K2) were weekly harvested during a 19-week long-period, starting from the fourth week after full bloom, until full maturity. At each sampling time, three replicates of 5 fruits each were dipped in the M.laxa conidial suspension or in distilled water, as negative control. The fruits were maintained at room temperature for 3 hours; afterwards, they were peeled with a scalpel; the peel was immediately frozen in liquid nitrogen and transferred to -80 °C until use. The degree of susceptibility of peach fruit to the pathogen was determined on 3 replicates of 20 fruits each, as percentage of infected fruits, after one week at 20 °C. Real time PCR analysis was performed to study the variation in expression of those genes encoding for the enzymes of the phenylpropanoid pathway (phenylalanine ammonia lyase (PAL), chalcone synthase (CHS), cinnamate 4-hydroxylase (C4H), leucoanthocyanidine reductase (LAR), hydroxycinnamoyl CoA quinate hydroxycinnamoyl transferase (HQT) and of the jasmonate pathway, such as lipoxygenase (LOX), both involved in the production of important defense compounds. Alteration in gene expression was monitored on fruit samples of a period encompassing the pit hardening stage and the corresponding temporary resistance to M.laxa infections, weekly, from the 6thto the 12th week after full bloom (AFB) inoculated with M. laxa or mock-inoculated. The data suggest a critical change in the expression level of the phenylpropanoid pathway from the 7th to the 8th week AFB; such change could be directly physiologically associated to the peach growth and it could indirectly determine the decrease of susceptibility of peach fruit to Monilinia rot during the subsequent weeks. To investigate on the transcriptome variation underneath the temporary loss of susceptibility of peach fruits to Monilinia rot, the microarray and the cDNA AFLP techniques were used. The samples harvested on the 8th week AFB (named S, for susceptible ones) and on the 12th week AFB (named R, for resistant ones) were compared, both inoculated or mock-inoculated. The microarray experiments were carried out at the University of Padua (Dept. of Environmental Agronomy and Crop Science), using the μPEACH1.0 microarray together with the suited protocols. The analysis showed that 30 genes (corresponding to the 0.6% of the total sequences (4806) contained in the μPeach1.0 microarray) were found up-regulated and 31 ( 0.6%) down regulated in RH vs. SH fruits. On the other hand, 20 genes (0.4%) were shown to be up-regulated and 13 (0.3%) down-regulated in the RI vs. SI fruit. No genes were found differentially expressed in the mock-inoculated resistant fruits (RH) vs. the inoculated resistant ones (RI). Among the up-regulated genes an ATP sulfurylase, an heat shock protein 70, the major allergen Pru P1, an harpin inducing protein and S-adenosylmethionine decarboxylase were found, conversely among the down-regulated ones, cinnamyl alcohol dehydrogenase, an histidine- containing phosphotransfer protein and the ferritin were found. The microarray experimental results and the data indirectly derived, were tested by Real Time PCR analysis. cDNA AFLP analysis was also performed on the same samples. 339 transcript derived fragments considered significant for Monilinia resistance, were selected, sequenced and classified. Genes potentially involved in cell rescue and defence were well represented (8%); several genes (12.1%) involved in the protein folding, post-transductional modification and genes (9.2%) involved in cellular transport were also found. A further 10.3% of genes were classified as involved in the metabolism of aminoacid, carbohydrate and fatty acid. On the other hand, genes involved in the protein synthesis (5.7%) and in signal transduction and communication (5.7%) were found. Among the most interesting genes found differentially expressed between susceptible and resistant fruits, genes encoding for pathogenesis related (PR) proteins were found. To investigate on the association of Monilinia resistance and PR biological function, the major allergen Pru P1 (GenBank accession AM493970) and its isoform (here named Pru P2), were expressed in heterologous system and in vitro assayed for their anti-microbial activity. The ribonuclease activity of the recombinant Pru P1 and Pru P2 proteins was assayed against peach total RNA. As the other PR10 proteins, they showed a ribonucleolytic activity, that could be important to contrast pathogen penetration. Moreover Pru P1 and Pru P2 recombinant proteins were checked for direct antimicrobial activity. No inhibitory effect of Pru P1 or Pru P2 was detected against the selected fungi.

Transkriptionskontrolle des nuoA-N Operons (NADH-Dehydrogenase I) von Escherichia coli

Das Elektronentransportsystem von E. coli enthält zwei verschiedene NADH-Dehydrogenasen. Die NADH-DehydrogenaseI (nuoA-N) koppelt im Gegensatz zur NADH-DehydrogenaseII die Oxidation von NADH an eine Protonentranslokation und trägt zur Energiekonservierung bei. Die NADH-DehydrogenaseI wird über die Promotoren P1 und P2 exprimiert und besitzt mehrere Bindestellen für verschiedene Regulatoren.Die separate Klonierung der Promotoren, lacZ-Fusionen, Inaktivierung von Transkriptionsfaktoren, sowie die Nutzung mutierter Regulatorbindestellen in vivo zeigen, dass P1 im wesentlichen die Expressionshöhe bestimmt und ist unter aeroben und anaeroben Bedingungen aktiv. P2 trägt in wesentlich geringerem Maße als P1 zur Expression des Enzyms bei. Er ist stark abhängig von ArcA und IHF. Beide Promotoren wirken nicht additiv.Unter anaeroben Bedingungen wird die Transkription von nuo durch das Zweikomponenten-System ArcB/A reprimiert. ArcA bindet unabhängig und mit unterschiedlicher Affinität an die beiden Bindestellen arc1 und arc2. Von den 8 ArcA-Konsensussequenzen führen nur Mutationen der Konsensussequenzen arc1ab in vitro zu verminderter Bindungsaffinität von ArcA an die Bindestelle arc1. Dieselben führen in vivo unter anaeroben Bedingungen zur Derepression des Promotors P1 bzw. P1+P2. Unter aeroben Bedingungen zeigen nur Mutationen in arc2 eine Derepression, die nicht durch ArcA vermittelt wird. Der veröffentliche ArcA-Konsensus scheint deshalb hier in dieser einfachen Form nicht gültig zu sein.

Anaerobic pathways in the Porifera: Strombine dehydrogenase, an opine dehydrogenase, from the sponge Suberites domuncula

The study presented here encompasses identification, analysis and characterization of the strombine dehydrogenase (StDH) from the sponge S. domuncula, on the gene and protein level. StDH is an opine dehydrogenase which is involved in opine production pathways found mainly in marine invertebrates. These anaerobic pathways are regarded as analogues to the classical anaerobic glycolytic pathway (lactate production pathway), which is predominant in vertebrates. The StDH was previously annotated as a tauropine dehydrogenase (TaDH) on the basis of its 68% identity with the TaDH protein from Halichondria japonica. Subsequent enzymatic assays showed that S. domuncula opine dehydrogenase is in fact strombine dehydrogenase which possesses specific characteristics not found in other proteins of the same family. It is described here for the first time the StDH gene in Eukaryotes. Two allelic variants have been identified which are present in the different specimens either as a homozygotic or a heterozygotic. Phylogenetic analyses supported with enzymatic assays indicate that S. domuncula StDH is only distantly related to the opine dehydrogenases from marine invertebrates. StDH showed that the protein is highly specific to glycine and inhibited by the substrate pyruvate. Furthermore, S. domunucla StDH has a dimeric structure (~75 kDa) which is not observed in so far described OpDHs that are monomeric proteins. This enzyme showed similarities to the OCD/mu-cristallyin protein family. Results showed that a sponge StDH is unusual enzyme that belongs to the independent enzyme class. In addition, expression studies revealed that the StDH is down-regulated with aeration. Immunohistology analyses showed high expression of the protein in almost all sponge cells. A strong accumulation of the enzyme was seen around the bacteria indicating that under aerobic conditions the bacteria might metabolize strombine (end product of the reaction). In conclusion, the data documented here shed new light on the anaerobic pathways in marine invertebrates. Potential mutual influences between bacteria and sponge are discussed as well. Hopefully, these results could have a small but important contribution to the better understanding of the evolution in the animal kingdom.

Loss of 15-Hydroxyprostaglandin Dehydrogenase Increases Prostaglandin E2 in Pancreatic Tumors

Prostaglandin E2 (PGE2) is a product of cyclooxygenase (COX) and PGE synthase (PGES) and deactivated by 15-hydroxyprostaglandin dehydrogenase (PGDH). Down-regulation of PGDH contributes to PGE2 accumulation in lung and colon cancers but has not been identified in pancreatic cancer.

Restoration of mutant cytochrome P450 reductase activity by external flavin

Cytochrome P450 oxidoreductase (POR) supplies electrons from NADPH to steroid and drug metabolizing reactions catalyzed by the cytochrome P450s located in endoplasmic reticulum. Mutations in human POR cause a wide spectrum of disease ranging from disordered steroidogenesis to sexual differentiation. Previously we and others have shown that POR mutations can lead to reduced activities of steroidogenic P450s CYP17A1, CYP19A1 and CYP21A1. Here we are reporting that mutations in the FMN binding domain of POR may reduce CYP3A4 activity, potentially influencing drug and steroid metabolism; and the loss of CYP3A4 activity may be correlated to the reduction of cytochrome b(5) by POR. Computational molecular docking experiments with a FMN free structural model of POR revealed that an external FMN could be docked in close proximity to the FAD moiety and receive electrons donated by NADPH. Using FMN supplemented assays we have demonstrated restoration of the defective POR activity in vitro.

The Combination of Interferon-Beta and HMG-CoA Reductase Inhibition in Multiple Sclerosis: Enthusiasm Lost too Soon?

Recent studies support the notion that statins, widely prescribed cholesterol-lowering agents, may target key elements in the immunological cascade leading to inflammation and tissue damage in the pathogenesis of multiple sclerosis (MS). Compelling experimental and observational clinical studies highlighted the possibility that statins may also exert immunomodulatory synergy with approved MS drugs, resulting in several randomized clinical trials testing statins in combination with interferon-beta (IFN-?). Some data, however, suggest that this particular combination may not be clinically beneficial, and might actually have a negative effect on the disease course in some patients with MS. In this regard, a small North American trial indicated that atorvastatin administered in combination with IFN-? may increase disease activity in relapsing-remitting MS. Although other trials did not confirm this finding, the enthusiasm for studies with statins dwindled. This review aims to provide a comprehensive overview of the completed clinical trials and reports of the interim analyses evaluating the combination of IFN-? and statins in MS. Moreover, we try to address the evident question whether usage of this combination routinely requires caution, since the number of IFN-?-treated MS patients receiving statins for lowering of cholesterol is expected to grow.

High aldehyde dehydrogenase activity identifies tumor-initiating and metastasis-initiating cells in human prostate cancer

Metastatic progression of advanced prostate cancer is a major clinical problem. Identifying the cell(s) of origin in prostate cancer and its distant metastases may permit the development of more effective treatment and preventive therapies. In this study, aldehyde dehydrogenase (ALDH) activity was used as a basis to isolate and compare subpopulations of primary human prostate cancer cells and cell lines. ALDH-high prostate cancer cells displayed strongly elevated clonogenicity and migratory behavior in vitro. More strikingly, ALDH-high cells readily formed distant metastases with strongly enhanced tumor progression at both orthotopic and metastatic sites in preclinical models. Several ALDH isoforms were expressed in human prostate cancer cells and clinical specimens of primary prostate tumors with matched bone metastases. Our findings suggest that ALDH-based viable cell sorting can be used to identify and characterize tumor-initiating and, more importantly perhaps, metastasis-initiating cells in human prostate cancer.

Altered heme catabolism by heme oxygenase-1 caused by mutations in human NADPH cytochrome P450 reductase

Human heme oxygenase-1 (HO-1) carries out heme catabolism supported by electrons supplied from the NADPH through NADPH P450 reductase (POR, CPR). Previously we have shown that mutations in human POR cause a rare form of congenital adrenal hyperplasia. In this study, we have evaluated the effects of mutations in POR on HO-1 activity. We used purified preparations of wild type and mutant human POR and in vitro reconstitution with purified HO-1 to measure heme degradation in a coupled assay using biliverdin reductase. Here we show that mutations in POR found in patients may reduce HO-1 activity, potentially influencing heme catabolism in individuals carrying mutant POR alleles. POR mutants Y181D, A457H, Y459H, V492E and R616X had total loss of HO-1 activity, while POR mutations A287P, C569Y and V608F lost 50-70% activity. The POR variants P228L, R316W and G413S, A503V and G504R identified as polymorphs had close to WT activity. Loss of HO-1 activity may result in increased oxidative neurotoxicity, anemia, growth retardation and iron deposition. Further examination of patients affected with POR deficiency will be required to assess the metabolic effects of reduced HO-1 activity in affected individuals.

Dihydropyrimidine dehydrogenase gene as a major predictor of severe 5-fluorouracil toxicity

The importance of polymorphisms in the dihydropyrimidine dehydrogenase (DPD) gene (DPYD) for the prediction of severe toxicity in 5-fluorouracil (5-FU) based chemotherapy has been controversially debated. As a key enzyme in the catabolism of 5-FU, DPD is the top candidate for pharmacogenetic studies on 5-FU toxicity, since a reduced DPD activity is thought to result in an increased half-life of the drug, and thus, an increased risk of toxicity. Here, we review the current knowledge on well-known and frequently studied DPYD variants such as the c.1905+1G>A splice site variant, as well as the recent discoveries of important functional variation in the noncoding regions of DPYD. We also outline future directions that are needed to further improve the risk assessment of 5-FU toxicity, in particular with respect to metabolic profiling and in the context of different combination therapeutic regimens, in which 5-FU is used today.

High expression of octamer-binding transcription factor 4A, prominin-1 and aldehyde dehydrogenase strongly indicates involvement in the initiation of lung adenocarcinoma resulting in shorter disease-free intervals

The increasing relevance of the cancer stem cell (CSC) hypothesis and the impact of CSC-associated markers in the carcinogenesis of solid tumours may provide potential prognostic implications in lung cancer. We propose that a collective genetic analysis of established CSC-related markers will generate data to better define the role of putative CSCs in lung adenocarcinoma (LAC).

High salt intake down-regulates colonic mineralocorticoid receptors, epithelial sodium channels and 11β-hydroxysteroid dehydrogenase type 2

Besides the kidneys, the gastrointestinal tract is the principal organ responsible for sodium homeostasis. For sodium transport across the cell membranes the epithelial sodium channel (ENaC) is of pivotal relevance. The ENaC is mainly regulated by mineralocorticoid receptor mediated actions. The MR activation by endogenous 11β-hydroxy-glucocorticoids is modulated by the 11β-hydroxysteroid dehydrogenase type 2 (11β-HSD2). Here we present evidence for intestinal segment specific 11β-HSD2 expression and hypothesize that a high salt intake and/or uninephrectomy (UNX) affects colonic 11β-HSD2, MR and ENaC expression. The 11β-HSD2 activity was measured by means of 3H-corticosterone conversion into 3H-11-dehydrocorticosterone in Sprague Dawley rats on a normal and high salt diet. The activity increased steadily from the ileum to the distal colon by a factor of about 3, an observation in line with the relevance of the distal colon for sodium handling. High salt intake diminished mRNA and protein of 11β-HSD2 by about 50% (p<0.001) and reduced the expression of the MR (p<0.01). The functionally relevant ENaC-β and ENaC-γ expression, a measure of mineralocorticoid action, diminished by more than 50% by high salt intake (p<0.001). The observed changes were present in rats with and without UNX. Thus, colonic epithelial cells appear to contribute to the protective armamentarium of the mammalian body against salt overload, a mechanism not modulated by UNX.

Uninephrectomy reduces 11β-hydroxysteroid dehydrogenase type 1 and type 2 concomitantly with an increase in blood pressure in rats

Renal allograft donors are at risk of developing hypertension. Here, we hypothesized that this risk is at least in part explained by an enhanced intracellular availability of 11β-hydroxyglucocorticoids due to an increased 11β-hydroxysteroid dehydrogenase type 1 enzyme (11β-HSD1), an intracellular prereceptor activator of biologically inactive 11-ketocorticosteroids in the liver, and/or a diminished 11β-hydroxysteroid dehydrogenase type 2 (11β-HSD2), an inactivator of 11β-hydroxyglucocorticoids in the kidney. To test this hypothesis, uninephrectomized (UNX) (n=9) and sham-operated (n=10) adult Sprague-Dawley rats were investigated. Mean arterial blood pressure and heart rate were measured continuously by telemetry for 6 days in week 5 after UNX. The mRNA of 11β-Hsd1 and 11β-Hsd2 in liver and kidney tissues were assessed by RT-PCR and the 11β-HSD activities were directly quantified in their corresponding tissues by determining the ratios of (tetrahydrocorticosterone+5α-tetrahydrocorticosterone)/tetrahydrodehydrocorticosterone ((THB+5α-THB)/THA) and of corticosterone/dehydrocorticosterone (B/A) by gas chromatography-mass spectrometry. The apparent total body activities of 11β-HSD1 and 11β-HSD2 were estimated using the urinary and plasma ratios of (THB+5α-THB)/THA and B/A. Mean arterial blood pressure was increased after UNX when compared with sham operation. Hepatic mRNA content of 11β-Hsd1 and hepatic, plasma, and urinary ratios of (THB+5α-THB)/THA were decreased after UNX, indicating diminished access of glucocorticoids to its receptors. In renal tissue, 11β-Hsd2 mRNA was reduced and B/A ratios measured in kidney, plasma, and urine were increased, indicating reduced 11β-HSD2 activity and enhanced access of glucocorticoids to mineralocorticoid receptors. Both 11β-HSD1 and 11β-HSD2 are downregulated after UNX in rats, a constellation considered to induce hypertension.