Clotting Properties of Onopordum tauricum (Willd.) Aqueous Extract in Milk of Different Species

Plant proteases used in cheesemaking are easily available and could increase the acceptability of cheeses, otherwise hindered by ethical issues (e.g., religions, dietary habits, aversion to genetically engineered food and food ingredients). The milk clotting potential ofOnopordum tauricum(Willd.) aqueous extract as an alternative to animal rennet was assessed for the first time in milk of different species (ewe, goat, cow). Among the aerial anatomical parts, i.e., receptacle, leaves, stems, and flowers, only the latter ones showed clotting properties. A response surface methodology (RSM) was used to explore the effects of three independent variables (temperature, pH, volume of coagulant) on the milk clotting activity (MCA) of the flower extract. A second-order polynomial model adequately described the experimental data and predicted a temperature value of 55 degrees C, a pH value of 4.9-5.7, and a volume of coagulant of 300-500 mu L (added to 5 mL of milk) as optimal conditions to maximize the MCA. At a 35 degrees C temperature and natural milk pH of 6.7-6.8, the estimated MCA of theO. tauricumextract was 72-87, 69-86, and 75-151, in goat's, ewe's, and cow's milk, respectively. In comparison, the MCA of calf rennet was 5.4-4.9, 3.3-14.7, and 4.9-16.7 times higher than that of the plant extract in goat's, ewe's, and cow's milk, respectively

Cancer cell metabolic plasticity allows resistance to NAMPT inhibition but invariably induces dependence on LDHA

Background: Inhibitors of nicotinamide phosphoribosyltransferase (NAMPT), the rate-limiting enzyme in NAD+ biosynthesis from nicotinamide, exhibit anticancer effects in preclinical models. However, continuous exposure to NAMPT inhibitors, such as FK866, can induce acquired resistance. Methods: We developed FK866-resistant CCRF-CEM (T cell acute lymphoblastic leukemia) and MDA MB231 (breast cancer) models, and by exploiting an integrated approach based on genetic, biochemical, and genome wide analyses, we annotated the drug resistance mechanisms. Results: Acquired resistance to FK866 was independent of NAMPT mutations but rather was based on a shift towards a glycolytic metabolism and on lactate dehydrogenase A (LDHA) activity. In addition, resistant CCRF-CEM cells, which exhibit high quinolinate phosphoribosyltransferase (QPRT) activity, also exploited amino acid catabolism as an alternative source for NAD+ production, becoming addicted to tryptophan and glutamine and sensitive to treatment with the amino acid transport inhibitor JPH203 and with L-asparaginase, which affects glutamine exploitation. Vice versa, in line with their low QPRT expression, FK866-resistant MDA MB231 did not rely on amino acids for their resistance phenotype. Conclusions: Our study identifies novel mechanisms of resistance to NAMPT inhibition, which may be useful to design more rational strategies for targeting cancer metabolism

Sviluppo di un metodo enzimatico di analisi per la discriminazione delle vie di utilizzo della vitamina B3 in differenti condizioni metaboliche

SVILUPPO DI UN METODO ENZIMATICO DI ANALISI PER LA DISCRIMINAZIONE DELLE VIE DI UTILIZZO DELLA VITAMINA B3 IN DIFFERENTI CONDIZIONI METABOLICHE Il NAD è una molecola chiave del metabolismo cellulare e il mantenimento dei suoi livelli intracellulari è garantito da un equilibrio tra processi enzimatici di sintesi e degradazione. La diminuzione dei livelli del coenzima è spesso associata all’insorgenza di patologie neurologiche e metaboliche ed è possibile prevenire o combattere tali disordini stimolando la rigenerazione del NAD mediante somministrazione del coenzima stesso e/o dei suoi precursori vitaminici. Al contrario, diminuire la biosintesi del NAD si sta rivelando una efficace terapia contro il cancro in quanto le cellule tumorali richiedono maggiori livelli di NAD per la loro sopravvivenza rispetto alle cellule normali. Il NAD può essere sintetizzato a partire dall’amminoacido triptofano o dalle tre forme della vitamina B3 attraverso quattro vie metaboliche distinte, ognuna delle quali contribuisce con diversa efficienza al mantenimento dei livelli del coenzima in base al tipo di cellula/tessuto e allo stato metabolico. La conoscenza dettagliata della distribuzione degli enzimi regolatori di ciascuna via nei vari organi e nelle diverse situazioni fisiopatologiche è stata finora limitata dalla mancanza di saggi adeguati alla determinazione dell’attività di tali enzimi in estratti cellulari. In questo lavoro abbiamo sviluppato un saggio che consente la determinazione simultanea delle attività dei quattro enzimi regolatori in matrici biologiche di vario tipo. L’applicazione del saggio in estratti cellulari preparati da diversi organi di topo e linee cellulari umane ha confermato l’utilità del saggio per la ricostruzione del pathway biosintetico del NAD operativo nelle varie cellule. I risultati ottenuti in cellule di carcinoma ovarico hanno permesso di proporre una nuova strategia chemioterapica, basata sull’inibizione contemporanea delle due vie metaboliche che maggiormente contribuiscono al mantenimento dei livelli del coenzima in questo tipo di cellule. La terapia sperimentata su modelli di topo ha mostrato una significativa riduzione della crescita del tumore e un significativo aumento della sopravvivenza.DEVELOPMENT OF ENZYMATIC ASSAY FOR THE DISCRIMINATION OF THE PATHWAY FOR THE VITAMIN B3 UTILIZATION IN DIFFERENT METABOLIC CONDITIONS NAD is a key metabolite in cellular metabolism and its homeostasis is the result of a delicate balance between NAD-cleaving and NAD-synthesizing reactions. The decrease of intracellular NAD levels is known to be the trigger of a variety of neurologic and metabolic disorders which can be prevented or contrasted by stimulating NAD salvage synthesis through the administration of NAD itself and/or its vitamin precursors. On the other hand, it has been proven that administration of inhibitors that restrain NAD biosynthesis is an effective tumor suppressing therapy because tumor cells require higher NAD levels to survive compared to normal cells. NAD can be synthesized starting from tryptophan or from the three forms of vitamin B3 through four distinct metabolic routes, which contribute to build up the overall coenzyme pool with different efficiencies and in various combinations depending on the different organism, tissue, cell and metabolic status. Knowledge of the distribution of the regulatory enzymes of each route in various tissues/organs and in different physiopathological conditions has been so far limited, essentially due to the lack of suitable assays for the determination of individual NAD-synthesizing enzyme activities in crude cell extracts. In this work we present an assay that allows the simultaneous determination of the activity of the four target enzymes in different biological samples. The application of the assay in crude extracts prepared from different mouse tissues and human cell lines demonstrated that the assay can be exploited to reconstruct the NAD biosynthetic machinery occurring in the various cell-types. Moreover, the results obtained on an ovarian carcinoma cell line allowed to propose a novel antitumoral strategy based on the combined inhibition of the target enzymes controlling the two metabolic routes that most contribute to the maintenance of NAD levels in this cell line. This combination therapy, tested in vivo on mouse models, resulted in a significant decrease of tumor growth and increase of mice survival

Sviluppo di un metodo enzimatico di analisi per la discriminazione delle vie di utilizzo della vitamina B3 in differenti condizioni metaboliche

SVILUPPO DI UN METODO ENZIMATICO DI ANALISI PER LA DISCRIMINAZIONE DELLE VIE DI UTILIZZO DELLA VITAMINA B3 IN DIFFERENTI CONDIZIONI METABOLICHE Il NAD è una molecola chiave del metabolismo cellulare e il mantenimento dei suoi livelli intracellulari è garantito da un equilibrio tra processi enzimatici di sintesi e degradazione. La diminuzione dei livelli del coenzima è spesso associata all’insorgenza di patologie neurologiche e metaboliche ed è possibile prevenire o combattere tali disordini stimolando la rigenerazione del NAD mediante somministrazione del coenzima stesso e/o dei suoi precursori vitaminici. Al contrario, diminuire la biosintesi del NAD si sta rivelando una efficace terapia contro il cancro in quanto le cellule tumorali richiedono maggiori livelli di NAD per la loro sopravvivenza rispetto alle cellule normali. Il NAD può essere sintetizzato a partire dall’amminoacido triptofano o dalle tre forme della vitamina B3 attraverso quattro vie metaboliche distinte, ognuna delle quali contribuisce con diversa efficienza al mantenimento dei livelli del coenzima in base al tipo di cellula/tessuto e allo stato metabolico. La conoscenza dettagliata della distribuzione degli enzimi regolatori di ciascuna via nei vari organi e nelle diverse situazioni fisiopatologiche è stata finora limitata dalla mancanza di saggi adeguati alla determinazione dell’attività di tali enzimi in estratti cellulari. In questo lavoro abbiamo sviluppato un saggio che consente la determinazione simultanea delle attività dei quattro enzimi regolatori in matrici biologiche di vario tipo. L’applicazione del saggio in estratti cellulari preparati da diversi organi di topo e linee cellulari umane ha confermato l’utilità del saggio per la ricostruzione del pathway biosintetico del NAD operativo nelle varie cellule. I risultati ottenuti in cellule di carcinoma ovarico hanno permesso di proporre una nuova strategia chemioterapica, basata sull’inibizione contemporanea delle due vie metaboliche che maggiormente contribuiscono al mantenimento dei livelli del coenzima in questo tipo di cellule. La terapia sperimentata su modelli di topo ha mostrato una significativa riduzione della crescita del tumore e un significativo aumento della sopravvivenza.DEVELOPMENT OF ENZYMATIC ASSAY FOR THE DISCRIMINATION OF THE PATHWAY FOR THE VITAMIN B3 UTILIZATION IN DIFFERENT METABOLIC CONDITIONS NAD is a key metabolite in cellular metabolism and its homeostasis is the result of a delicate balance between NAD-cleaving and NAD-synthesizing reactions. The decrease of intracellular NAD levels is known to be the trigger of a variety of neurologic and metabolic disorders which can be prevented or contrasted by stimulating NAD salvage synthesis through the administration of NAD itself and/or its vitamin precursors. On the other hand, it has been proven that administration of inhibitors that restrain NAD biosynthesis is an effective tumor suppressing therapy because tumor cells require higher NAD levels to survive compared to normal cells. NAD can be synthesized starting from tryptophan or from the three forms of vitamin B3 through four distinct metabolic routes, which contribute to build up the overall coenzyme pool with different efficiencies and in various combinations depending on the different organism, tissue, cell and metabolic status. Knowledge of the distribution of the regulatory enzymes of each route in various tissues/organs and in different physiopathological conditions has been so far limited, essentially due to the lack of suitable assays for the determination of individual NAD-synthesizing enzyme activities in crude cell extracts. In this work we present an assay that allows the simultaneous determination of the activity of the four target enzymes in different biological samples. The application of the assay in crude extracts prepared from different mouse tissues and human cell lines demonstrated that the assay can be exploited to reconstruct the NAD biosynthetic machinery occurring in the various cell-types. Moreover, the results obtained on an ovarian carcinoma cell line allowed to propose a novel antitumoral strategy based on the combined inhibition of the target enzymes controlling the two metabolic routes that most contribute to the maintenance of NAD levels in this cell line. This combination therapy, tested in vivo on mouse models, resulted in a significant decrease of tumor growth and increase of mice survival

Clotting Properties of Onopordum tauricum (Willd.) Aqueous Extract in Milk of Different Species

Plant proteases used in cheesemaking are easily available and could increase the acceptability of cheeses, otherwise hindered by ethical issues (e.g., religions, dietary habits, aversion to genetically engineered food and food ingredients). The milk clotting potential of Onopordum tauricum (Willd.) aqueous extract as an alternative to animal rennet was assessed for the first time in milk of different species (ewe, goat, cow). Among the aerial anatomical parts, i.e., receptacle, leaves, stems, and flowers, only the latter ones showed clotting properties. A response surface methodology (RSM) was used to explore the effects of three independent variables (temperature, pH, volume of coagulant) on the milk clotting activity (MCA) of the flower extract. A second-order polynomial model adequately described the experimental data and predicted a temperature value of 55 °C, a pH value of 4.9–5.7, and a volume of coagulant of 300–500 μL (added to 5 mL of milk) as optimal conditions to maximize the MCA. At a 35 °C temperature and natural milk pH of 6.7–6.8, the estimated MCA of the O. tauricum extract was 72–87, 69–86, and 75–151, in goat’s, ewe’s, and cow’s milk, respectively. In comparison, the MCA of calf rennet was 5.4–4.9, 3.3–14.7, and 4.9–16.7 times higher than that of the plant extract in goat’s, ewe’s, and cow’s milk, respectively

Exploratory study on the occurrence and dynamics of yeast-mediated nicotinamide riboside production in craft beer

none8noIn this study, nicotinamide riboside (NR), a form of vitamin B3, and its precursors nicotinamide mononucleotide (NMN) and NAD+ were quantitated by a fluorometric assay in ten commercial craft beers and throughout the fermentation process of laboratory-scale beer. All tested beers from different commercial S. cerevisiae strains contained NAD+ (ranging from 1.10 μM to 17.80 μM). NR concentrations ranged from 0.48 to 3.25 μM, while the content of NMN was approximately 0.9 μM. NR and NMN were only present in beers produced with Saccharomyces cerevisiae strain US-05. Data from laboratory-scale beer productions using S. cerevisiae strain US-05 showed that the addition of hops [amarillo (alpha acid: 9.0%) (4 g/L) and centennial (alpha acid: 8.5%) (4 g/L) varieties] at the 9th day of fermentation significantly increased NR production up to 6.11 μM at the end of a typical fermentation of 21 days, and up to 11.42 μM at 45 days of fermentation. The rapid increase in NR formation only occurred if both hops and yeast were present, and the burst was also confirmed in fermentations trials performed with S. cerevisiae strain CBS1171T and by replacing wort with YPD medium. This study can serve as baseline for further research on yeast-hop interaction.noneCristiana Garofalo, Riccardo Sabbatini, Federica Zamporlini, Gabriele Minazzato, Ilario Ferrocino, Lucia Aquilanti, Nadia Raffaelli, Andrea OsimaniGarofalo, Cristiana; Sabbatini, Riccardo; Zamporlini, Federica; Minazzato, Gabriele; Ferrocino, Ilario; Aquilanti, Lucia; Raffaelli, Nadia; Osimani, Andre

Simultaneous quantitation of nicotinamide riboside, nicotinamide mononucleotide and nicotinamide adenine dinucleotide in milk by a novel enzyme-coupled assay

Nicotinamide riboside, the most recently discovered form of vitamin B3, and its phosphorylated form nicotinamide mononucleotide, have been shown to be potent supplements boosting intracellular nicotinamide adenine dinucleotide (NAD) levels, thus preventing or ameliorating metabolic and mitochondrial diseases in mouse models. Here we report for the first time on the simultaneous quantitation of nicotinamide riboside, nicotinamide mononucleotide and NAD in milk by means of a fluorometric, enzyme-coupled assay. Application of this assay to milk from different species revealed that the three vitamers were present in human and donkey milk, while being selectively distributed in the other milks. Human milk was the richest source of nicotinamide mononucleotide. Overall, the three vitamers accounted for a significant fraction of total vitamin B3 content. Pasteurization did not affect the bovine milk content of nicotinamide riboside, whereas UHT processing fully destroyed the vitamin. In human milk, NAD levels were significantly affected by the lactation time