Indagini sulla produzione di tannasi extracellulari in specie di Fusarium: aspetti fisiologici e biochimici

ANNO ACCADEMICO 2005/2006 Candidato: Francesca Tardelli Relatore: D.ssa Susanna Pecchia Correlatore: D.ssa Lucia Guidi Corso di Laurea Magistrale in Biotecnologie Vegetali e Microbiche Tesi Indagini sulla produzione di tannasi extracellulari in specie di Fusarium: aspetti fisiologici e biochimici La produzione di tannasi extracellulari è stata valutata in 30 isolati di Fusarium appartenenti a differenti sezioni: 4 isolati di F. oxysporum e 9 forme speciali della medesima specie, appartenenti alla sezione Elegans; 2 isolati di F. anthophylum, 5 di F. moniliforme, 5 di F. proliferatum ed 1 isolato di F. sacchari, appartenenti alla sezione Liseola; infine, 1 isolato di F. solani della sezione Martiella e 3 isolati di F. sporotrichioides della sezione Sporotrichiella utilizzati come di specie controllo. Le indagini fisiologiche si sono basate sull’analisi di dati precedentemente ottenuti a seguito della realizzazione di prove in piastra, condotte su substrato contenente acido tannico come unica fonte di carbonio, eseguite su un range di 197 isolati di Fusarium spp. L’attività dell’enzima tannasi, responsabile del processo di scissione dell’acido tannico in acido gallico e glucosio, è stata valutata mediante misurazione del fenomeno di chiarificazione del substrato di coltura considerato, ossia mediate la misurazione dell’alone di chiarificazione prodotto dal fungo. L’analisi dei dati fisiologici ha consentito di individuare caratteristiche differenziali tra due delle principali sezioni del genere Fusarium, Elegans e Liseola. E’ stato possibile osservare che il tempo in corrispondenza del quale veniva raggiunto il massimo incremento nella produzione dell’alone differiva tra le due sezioni. All’interno della sezione Elegans, inoltre, gli isolati di F. oxysporum hanno manifestato una certa eterogeneità di comportamento mentre le forme speciali di F. oxysporum hanno evidenziato un comportamento più omogeneo. Nella sezione Liseola, invece, sono gli isolati di F. moniliforme a manifestare una variabilità maggiore per quanto concerne gli incrementi dell’alone. Tuttavia essi mostravano una certa omogeneità relativa al tempo necessario al raggiungimento del massimo incremento dell’alone di chiarificazione. L’analisi dei dati fisiologici ha consentito, inoltre, di effettuare una scelta degli isolati appartenenti alle due sezioni oggetto dell’indagine che, tra tutti, manifestavano le migliori capacità di chiarificazione del substrato. Su tali isolati si è proceduto, quindi, ad effettuare indagini biochimiche relative all’analisi dell’attività enzimatica specifica della tannasi, che hanno evidenziato, nella maggior parte delle specie di Fusarium, un’attività per lo più ridotta o inesistente. Il confronto dei risultati ottenuti dalle indagini fisiologiche e biochimiche ha consentito di evidenziare che, in generale, non esiste una correlazione diretta tra i due tipi di analisi effettuate. Solo per alcuni isolati (F. oxysporum 3618, F. moniliforme 24 e F. sacchari 3685) che presentavano in tempi relativamente brevi incrementi massimi dell’alone di chiarificazione sono stati rilevati anche livelli di attività specifica dell’enzima tannasi significativamente superiori a tutti gli altri. E’ stato, infine, valutato il peso molecolare della tannasi negli isolati 6363 di F. moniliforme e 465 di F. solani, mediante elettroforesi su gel di poliacrilammide in condizioni denaturanti (SDSPAGE). Anche per questa indagine sono stati analizzati, come controlli di riferimento, i tre isolati di F. sporotrichioides, esempio di mancata attività tannasica in piastra. I risultati ottenuti ci hanno permesso di ipotizzare che nei due isolati considerati la tannasi sia multimerica e costituita da almeno due subunità. In conclusione, le indagini condotte hanno permesso di caratterizzare, per la prima volta, diverse specie di Fusarium, per la capacità di utilizzare l’acido tannico come unica fonte di carbonio e costituiscono una base di partenza per ulteriori studi ecologici, fisiologici e molecolari della tannasi nel genere Fusarium

Pre- and post-harvest factors influencing quality, organoleptics, nutrition properties and physiology of fresh-cut fruits

Food is anylonger considered just a source of nutrients indeed the protective and functional meaning it has assumed during times is strictly linked to the concepts of maintenance and implementation of human well-being. Oxygen hides in itself the concept of life underlying the fact that most of the organisms cannot live in absence of oxygen. However, its use is also associated with the generation of reactive oxygen species (ROS) that are the radicals generated by the organism known to be responsible for the oxidative damage of biological macromolecules such as DNA, carbohydrates and proteins. Biological damages prevention and detoxification processes are enzymatically or non-enzymatically antioxidant systems that can be activated by the organism determining a significative delay or inhibition of ROSs. These antioxidants substances are highly concentrated in plant tissues and represent the preferential target for oxidants and free radicals thus exerting a cells protective role against oxidative stress, for both humans and animals. Moreover, they are very important in food preservation, slowing down processes like deterioration or colour loss due to oxidation processes. The most preeminent representative compounds exherting an antioxidant activity in human diet are ascorbate (vitamin C), tocopherols (vitamin E), carotenoids and flavonoids. Ready-to-eat foods like fruit and vegetables, are quick products, time saving, fresh, highly nutritious and low priced, all reasons why they are considered products with several added values, full of benefits. However, in respect to entire fruits or vegetables, fresh-cut products have a shorter shelf-life due to the higher metabolic activity which is stimulated by a tissues’ wounding. In fact, even if these kinds of products undergo a minimal processing, operations like peeling, cutting, slicing, are the causes of mechanical tissue injuries that are responsible for a reduced life. Moreover, the recent awareness of the existence of a large group of organic bioactive compounds, that are not strictly required in the diet but that can promote good health and prevent diseases, led consumers to look for good quality parameters such as texture, flavor and health-promoting compounds, that are all considered important internal quality attributes. Fruit quality, as well as vegetable one, can be improved by accurate selection on genotype, agricultural practices, such as water management, fertilization, and environmental conditions, in terms of light exposition, temperature and air composition. Because of the fact that there are several factors belonging to both the periods before and after harvest that are involved in fruit development and in fruit quality determination, the general aim of the studies conducted was the evaluation of the influence of pre- and post-harvest factors over quality, organoleptics, nutrition properties and physiology of fresh-cut products made from apples and kiwifruits. Regarding apple fruit, a study evaluating the effects of the cold-storage on nutritional and organoleptics quality of slices prepared from Red Delicious and Granny Smith apples was carried out. In addition to, evaluations on the influence of ClO2 and/or ascorbic acid treatments as preserving strategies for fresh-cut products quality improvement were also examined. The study led to the conclusion that qualitative intrinsic characters of whole fruits as well as sanitizing and antioxidant treatments used for fresh-cut apple fruits had a statistically significant influence on product shelf life and aspect, in terms of flesh firmness, flavor, tissue browning and phytochemical content. From a different perspective, the effect of 1-MCP treatment and two different holding times for short-term post-controlled air storage (PCAAS) treatment were considered to evaluate quality retaining capacity and physiology of fresh-cut slices made from apples belonging to AmbrosiaTM cultivar. The most dramatic effect of the PCAAS treatments was to reduce the accumulations of soluble phenolics, which is likely the reason that o-quinone accumulations were also inhibited in treated fruit. The consequent reduction in browning potential may be the explanation as to why PCAAS treatment has been shown to reduce fresh apple slice browning in previous work. In this study polyphenol oxidase (PPO) activity was not detected in slices treated with calcium ascorbate (CaAsc). In addition, bioaccessibility in the small intestine of dietary polyphenols was estimated considering four different apple varieties. The study was conducted in order to quantify the total oxyradical scavenging of the original samples compared to the potentially bioavailable residues of the enzymatic in vitro digestion process. This part of the study also focused on the determination of dietary fiber content in the attempt to understand the amount of polyphenols associated to the indigestible fraction. Significant differences in reducing capacity (total phenolics content) of the four cultivars were observed when intact apples were measured, however, digestion of the apples resulted in significantly reduced reducing capacity in the dialyzable soluble fraction of all cultivars. Differences between the cultivars were not great for reducing capacity after digestion, suggesting that breeding for higher phenolics may not lead to significantly healthier outcomes for consumers. Similar results were seen in the peroxyl radical (TOSC) capacity analyses, which were again highly correlated to phenolic contents. Further analysis determined that one of the mechanisms for loss of soluble phenolics could be attributed to binding with insoluble fiber during digestion. In regards to kiwifruit, the evaluation of organoleptics and nutritional characteristics at different times of cold storage was performed. Moreover, kiwifruit quality parameters were analyzed after minimal processing in order to understand the effect of the storage conditions and of the light or shade incidence – dependent on fruit canopy position - on the shelf life of fresh-cut kiwifruit. Even when stored for the longest period (150 d) kiwifruit preserved good quality parameters. In the attempt to evaluate together the influences of storage time, light influence and minimal processing we observed that when processed as fresh-cut over both storage periods, 75 and 150 d, kiwifruits were characterized by a decrease in organoleptic parameters that was evident also for nutritional aspects only after 24 hours from minimal processing. The best quality and nutritional retention was found for fruits grown in light condition after 75 d of cold storage. Differently, when vitamin C content was considered it was possible to reach the conclusion that the longest the storage period the worse the influence on this molecule retention, much more than the impact of light/shade conditions. Sunlight fruit exposure resulted to have a great influence particularly on enzymatic activities directly involved in softening processes. In conclusion, data suggests that the quality of kiwifruits and their capability to be stored as fresh-cut products is influenced by pre-harvest factors. The answer is complicated by the fact that organoleptics and nutritional indices did not respond in the same manner to these factors

Absence of Adiponutrin (PNPLA3) and Monoacylglycerol Lipase Synergistically Increases Weight Gain and Aggravates Steatohepatitis in Mice

Altered lipid metabolic pathways including hydrolysis of triglycerides are key players in the pathogenesis of nonalcoholic fatty liver disease (NAFLD). Whether adiponutrin (patatin-like phospholipase domain containing protein-3-PNPLA3) and monoacylglycerol lipase (MGL) synergistically contribute to disease progression remains unclear. We generated double knockout (DKO) mice lacking both Mgl and Pnpla3; DKO mice were compared to Mgl-/- after a challenge by high-fat diet (HFD) for 12 weeks to induce steatosis. Serum biochemistry, liver transaminases as well as histology were analyzed. Fatty acid (FA) profiling was assessed in liver and adipose tissue by gas chromatography. Markers of inflammation and lipid metabolism were analyzed. Bone marrow derived macrophages (BMDMs) were isolated and treated with oleic acid. Combined deficiency of Mgl and Pnpla3 resulted in weight gain on a chow diet; when challenged by HFD, DKO mice showed increased hepatic FA synthesis and diminished beta-oxidation compared to Mgl-/-.DKO mice exhibited more pronounced hepatic steatosis with inflammation and recruitment of immune cells to the liver associated with accumulation of saturated FAs. Primary BMDMs isolated from the DKO mice showed increased inflammatory activities, which could be reversed by oleic acid supplementation. Pnpla3 deficiency aggravates the effects of Mgl deletion on steatosis and inflammation in the liver under HFD challenge

Monoacylglycerol Lipase Inhibition Protects From Liver Injury in Mouse Models of Sclerosing Cholangitis

Background and Aims Monoacylglycerol lipase (MGL) is the last enzymatic step in triglyceride degradation, hydrolyzing monoglycerides into glycerol and fatty acids (FAs) and converting 2-arachidonoylglycerol into arachidonic acid, thus providing ligands for nuclear receptors as key regulators of hepatic bile acid (BA)/lipid metabolism and inflammation. We aimed to explore the role of MGL in the development of cholestatic liver and bile duct injury in mouse models of sclerosing cholangitis, a disease so far lacking effective pharmacological therapy. Approach and Results To this aim we analyzed the effects of 3,5-diethoxycarbonyl-1,4-dihydrocollidine (DDC) feeding to induce sclerosing cholangitis in wild-type (WT) and knockout (MGL(-/-)) mice and tested pharmacological inhibition with JZL184 in the multidrug resistance protein 2 knockout (Mdr2(-/-)) mouse model of sclerosing cholangitis. Cholestatic liver injury and fibrosis were assessed by serum biochemistry, liver histology, gene expression, and western blot characterization of BA and FA synthesis/transport. Moreover, intestinal FAs and fecal microbiome were analyzed. Transfection and silencing were performed in Caco2 cells. MGL(-/-) mice were protected from DDC-induced biliary fibrosis and inflammation with reduced serum liver enzymes and increased FA/BA metabolism and beta-oxidation. Notably, pharmacological (JZL184) inhibition of MGL ameliorated cholestatic injury in DDC-fed WT mice and protected Mdr2(-/-) mice from spontaneous liver injury, with improved liver enzymes, inflammation, and biliary fibrosis. In vitro experiments confirmed that silencing of MGL decreases prostaglandin E-2 accumulation in the intestine and up-regulates peroxisome proliferator-activated receptors alpha and gamma activity, thus reducing inflammation. Conclusions Collectively, our study unravels MGL as a metabolic target, demonstrating that MGL inhibition may be considered as potential therapy for sclerosing cholangitis

Hepatic Medicine: Evidence and Research / PNPLA3 expression and its impact on the liver: current perspectives

A single-nucleotide polymorphism occurring in the sequence of the human patatin-like phospholipase domain-containing 3 gene (PNPLA3), known as I148M variant, is one of the best characterized and deeply investigated variants in several clinical scenarios, because of its tight correlation with increased risk for developing hepatic steatosis and more aggressive part of the disease spectrum, such as nonalcoholic steatohepatitis, advanced fibrosis and cirrhosis. Further, the I148M variant is positively associated with alcoholic liver diseases, chronic hepatitis Crelated cirrhosis and hepatocellular carcinoma. The native gene encodes for a protein that has not yet a fully defined role in liver lipid metabolism and, according to recent observations, seems to be divergently regulated among distinct liver cells type, such as hepatic stellate cells. Therefore, the aim of this review is to collect the latest data regarding PNPLA3 expression in human liver and to analyze the impact of its genetic variant in human hepatic pathologies. Moreover, a description of the current biochemical and metabolic data pertaining to PNPLA3 function in both animal models and in vitro studies is summarized to allow a better understanding of the relevant pathophysiological role of this enzyme in the progression of hepatic diseases.(VLID)488575

Nuclear Receptor Regulation of Aquaglyceroporins in Metabolic Organs

Nuclear receptors, such as the farnesoid X receptor (FXR) and the peroxisome proliferator-activated receptors gamma and alpha (PPAR-, -), are major metabolic regulators in adipose tissue and the liver, where they govern lipid, glucose, and bile acid homeostasis, as well as inflammatory cascades. Glycerol and free fatty acids are the end products of lipid droplet catabolism driven by PPARs. Aquaporins (AQPs), a family of 13 small transmembrane proteins, facilitate the shuttling of water, urea, and/or glycerol. The peculiar role of AQPs in glycerol transport makes them pivotal targets in lipid metabolism, especially considering their tissue-specific regulation by the nuclear receptors PPAR and PPAR. Here, we review the role of nuclear receptors in the regulation of glycerol shuttling in liver and adipose tissue through the function and expression of AQPs.(VLID)471770

Effect of Chlorine Dioxide and Ascorbic Acid on Enzymatic Browning and Shelf Life of Fresh-Cut Red Delicious and Granny Smith Apples

In this work, we tested the hypothesis that ascorbic acid (AA) reduces browning of fresh-cut apples (Red Delicious, RD, and Granny Smith, GS), and we investigated the impact of AA on phenylpropanoid metabolism of RD and GS. Apple slices were dipped in a solution of 100mg/L of chlorine dioxide (ClO2) and ClO2+3% AA and stored at 4C for 96h. Flesh firmness, solid soluble content and browning index, total phenols and flavonoids, and the activity of peroxidase and polyphenol oxidase were monitored upon storage (0, 48 and 96h). Our results demonstrated that GS is less sensitive to browning and thus more suitable for minimally processed produce. Ascorbate reduces the browning index also in RD, a cultivar largely appreciated by consumers but more prone to browning. AA likely contrasts browning appearance by interacting with peroxidase and polyphenol oxidase and/or promoting the regeneration of phenols and flavonoids. Practical Applications: Browning of fresh-cut apple is one of the main problems that limit the shelf life of this type of produce. Given that this produce is highly appreciated by consumers, different antibrowning treatments have been tested to extend the shelf life of fresh-cut apple. We found that treatment with 100mg/L of ClO2+3% of ascorbic acid significantly reduces the browning appearance in apple slices. Browning was also reduced in Red Delicious cultivar that is more prone than Granny Smith to this phenomenon, but that is highly appreciated by consumers

AQP3 is regulated by PPARγ and JNK in hepatic stellate cells carrying PNPLA3 I148M

Abstract Aquaglyceroporins (AQPs) allow the movement of glycerol that is required for triglyceride formation in hepatic stellate cells (HSC), as key cellular source of fibrogenesis in the liver. The genetic polymorphism I148M of the patatin-like phospholipase domain-containing 3 (PNPLA3) is associated with hepatic steatosis and its progression to steatohepatitis (NASH), fibrosis and cancer. We aimed to explore the role of AQP3 for HSC activation and unveil its potential interactions with PNPLA3. HSC were isolated from human liver, experiments were performed in primary HSC and human HSC line LX2. AQP3 was the only aquaglyceroporin present in HSC and its expression decreased during activation. The PPARγ agonist, rosiglitazone, recovered AQP3 expression also in PNPLA3 I148M carrying HSC. When PNPLA3 was silenced, AQP3 expression increased. In liver sections from patients with NASH, the decreased amount of AQP3 was proportional to the severity of fibrosis and presence of the PNPLA3 I148M variant. In PNPLA3 I148M cells, the blockade of JNK pathway upregulated AQP3 in synergism with PPARγ. In conclusion, we demonstrated profound reduction of AQP3 in HSC carrying the PNPLA3 I148M variant in parallel to decreased PPARγ activation, which could be rescued by rosiglitazone and blockade of JNK

Molecular and biochemical responses to wounding in mesocarp of ripe peach (Prunus persica L. Batsch) fruit

The physiological and molecular responses of ripe fruit to wounding were evaluated in two peach (Prunus persica) varieties ('Glohaven', GH, melting and 'BigTop', BT, slow melting nectarine) by comparing mesocarp samples from wedges (as in minimal processing) and whole fruit as the control. Slight differences between the two varieties were detected in terms of ethylene production, whereas total phenol and flavonoid concentrations, and PPO and POD enzyme activities showed a general increase in wounded GH but not in BT. This was associated with the better appearance of the BT wedges at the end of the experimental period (72h). Microarray (genome-wide μPEACH3.0) analysis revealed that a total number of 2218 genes were differentially expressed (p<0.01, log2 fold change expression ratio >1 or <-1) in GH 24h after wounding compared to the control. This number was much lower (1208) in BT. According to the enrichment analysis, cell wall, plasma membrane, response to stress, secondary metabolic processes, oxygen binding were the GO categories over-represented among the GH up-regulated genes, whereas plasma membrane and response to endogenous stimulus were the categories over-represented among the down-regulated genes. Only 32 genes showed a common expression trend in the two varieties 24h after wounding, whereas a total of 512 genes (with highly represented transcription factors), displayed opposite behavior. Quantitative RT-PCR analysis confirmed the microarray data for 18 out of a total of 20 genes selected. Specific WRKY, AP2/ERF and HSP20 genes were markedly up-regulated in wounded GH, indicating the activation of regulatory and signaling mechanisms probably related to different hormone categories. Compared to BT, the expression of specific genes involved in phenylpropanoid and triterpenoid biosynthetic pathways showed a more pronounced induction in GH, highlighting the difference between the two peach varieties in terms of molecular responses to wounding in the mesocarp tissue. © 2013 Elsevier B.V