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Marta Cascante

Publications and source records attributed to Marta Cascante.

At least 19 recordsLinked to original sources
PubMed2003

Metabolic strategy of boar spermatozoa revealed by a metabolomic characterization.

Metabolomic characteristics in boar spermatozoa were studied using [1,2-(13)C(2)]glucose and mass isotopomer analysis. In boar spermatozoa, glycolysis was the main pathway of glucose utilization producing lactate/pyruvate, whereas no gluconeogenesis was seen. Slight glycogen synthesis through the direct pathway and some incorporation of pyruvate into the Krebs cycle also took place. Neither RNA ribose-5-phosphate nor fatty acid synthesis from glucose occurred despite the detection of pyruvate dehydrogenase activity. In contrast to the known metabolic activities in dog sperm, boar spermatozoa have low levels of energy production and biosynthetic activities suggesting two different metabolic profiles for the two different phenotypes.

Animals↗
PubMed2003

Glucose conversion by multiple pathways in brain extract: theoretical and experimental analysis.

Experimental and model studies were performed to characterize the flux of glucose metabolism and the sharing of glucose-6-phosphate (Glu6P) by the upper parts of glycolytic and pentosephosphate pathways in the brain extract. A mathematical model based upon the kinetic equations of the individual enzymes was evaluated to fit the experimental data. Glucose is converted to glucose-6-phosphate by hexokinase that controls almost exclusively the glucose metabolism. Experiments showed that this crossroad-metabolite was shared between glycolysis and pentosephosphate pathway in the brain extract in a ratio of 1.5:1. This ratio was favorable to the pentosephosphate pathway by the addition of high excess of exogenous glucose-6-phosphate dehydrogenase, standardly used for the activity assay of hexokinase, but still a significant part (17+/-3%) of the common intermediate was converted into the direction of glycolysis. Stimulation of glucose-6-phosphate formation via moderate (30-50%) increase of hexokinase activity by adding exogenous hexokinase or tubulin resulted in the slight increase of the relative flux into direction of glycolysis. The model correctly described all of these observations. However, when the activity of hexokinase was doubled with exogenous enzyme, significantly less glucose-6-phosphate was converted into direction of glycolysis than predicted. This discrepancy shows that the system did not behave in this case as an ideal one, which could be due to the formation of distinct pools for the intermediate.

Animals↗
PubMed2003

Characterization of the transit and transition times for a pathway unit of Michaelis-Menten mechanism.

The transit time and a characteristic time constant for the transition time are formulated for a pathway unit of Michaelis-Menten mechanism in metabolic chains with mass-action-type dependence. The pathway unit in a chain is assigned to each metabolite and two consecutive Michaelis-Menten-type reactions associated with it. All the relevant functions such as control coefficients, elasticities and transit time for the pathway unit are expressed in terms of the flux J and other system parameters. The transition time is characterized by a time constant for a first-order system expressed as the derivative of the total concentration of the internal metabolite with respect to the flux J.

Computer Simulation↗
PubMed2003

Multicriteria optimization of biochemical systems by linear programming: application to production of ethanol by Saccharomyces cerevisiae.

In this study we present a method for simultaneous optimization of several metabolic responses of biochemical pathways. The method, based on the use of the power law formalism to obtain a linear system in logarithmic coordinates, is applied to ethanol production by Saccharomyces cerevisiae. Starting from an experimentally based kinetic model, we translated it to its power law equivalent. With this new model representation, we then applied the multiobjective optimization method. Our intent was to maximize ethanol production and minimize each of the internal metabolite concentrations. To ensure cell viability, all optimizations were carried out under imposed constraints. The different solutions obtained, which correspond to alternative patterns of enzyme overexpression, were implemented in the original model. We discovered few discrepancies between the S-system-optimized steady state and the corresponding optimized state in the original kinetic model, thus demonstrating the suitability of the S-system representation as the basis for the optimization procedure. In all optimized solutions, the ATP level reached its maximum and any increase in its activity positively affected the optimization process. This work illustrates that in any optimization study no single criteria is of general application being the multiobjective and constrained task the proper way to address it. It is concluded that the proposed multiobjective method can serve to carry out, in a single study, the general pattern of behavior of a given metabolic system with regard to its control and optimization.

Algorithms↗
PubMed2003

Defective RNA ribose synthesis in fibroblasts from patients with thiamine-responsive megaloblastic anemia (TRMA).

Fibroblasts from patients with thiamine-responsive megaloblastic anemia (TRMA) syndrome with diabetes and deafness undergo apoptotic cell death in the absence of supplemental thiamine in their cultures. The basis of megaloblastosis in these patients has not been determined. Here we use the stable [1,2-13C2]glucose isotope-based dynamic metabolic profiling technique to demonstrate that defective high-affinity thiamine transport primarily affects the synthesis of nucleic acid ribose via the nonoxidative branch of the pentose cycle. RNA ribose isolated from TRMA fibroblasts in thiamine-depleted cultures shows a time-dependent decrease in the fraction of ribose derived via transketolase, a thiamine-dependent enzyme in the pentose cycle. The fractional rate of de novo ribose synthesis from glucose is decreased several fold 2 to 4 days after removal of thiamine from the culture medium. No such metabolic changes are observed in wild-type fibroblasts or in TRMA mutant cells in thiamine-containing medium. Fluxes through glycolysis are similar in TRMA versus control fibroblasts in the pentose and TCA cycles. We conclude that reduced nucleic acid production through impaired transketolase catalysis is the underlying biochemical disturbance that likely induces cell cycle arrest or apoptosis in bone marrow cells and leads to the TRMA syndrome in patients with defective high-affinity thiamine transport.

Anemia, Megaloblastic↗
PubMed2003

The stable isotope-based dynamic metabolic profile of butyrate-induced HT29 cell differentiation.

Stable isotope-based dynamic metabolic profiling is applied in this paper to elucidate the mechanism by which butyrate induces cell differentiation in HT29 cells. We utilized butyrate-sensitive (HT29) cells incubated with [1,2-13C2]glucose or [1,2-13C2]butyrate as single tracers to observe the changes in metabolic fluxes in these cells. In HT29 cells, increasing concentrations of butyrate inhibited glucose uptake, glucose oxidation, and nucleic acid ribose synthesis in a dose-dependent fashion. Glucose carbon utilization for de novo fatty acid synthesis and tricarboxylic acid cycle flux was replaced by butyrate. We also demonstrated that these changes are not present in butyrate-resistant pancreatic adenocarcinoma MIA cells. The results suggest that the mechanism by which colon carcinoma cells acquire a differentiated phenotype is through a replacement of glucose for butyrate as the main carbon source for macromolecule biosynthesis and energy production. This provides a better understanding of cell differentiation through metabolic adaptive changes in response to butyrate in HT29 cells, demonstrating that variations in metabolic pathway substrate flow are powerful regulators of tumor cell proliferation and differentiation.

Acetyl Coenzyme A↗
PubMed2003

Antiproliferative effect of antioxidant polyphenols from grape in murine Hepa-1c1c7.

BACKGROUND: Grapes and wine contain high concentrations of polyphenolic compounds. Although their cancer protective effect has been well documented, their activity as anticarcinogens should be cautiously considered since the molecular bases of action and their applicability to human cancer prevention are still unclear. AIM OF THE STUDY: We studied the antioxidant/antiradical activity and the antiproliferative effect in vitro of different polyphenolic mixtures, extracted from grapes and fractionated through RP-HPLC. METHODS: The polyphenolic fractions were chemically characterized and their antioxidant/antiradical activity was determined by the DPPH assay. Mouse hepatoma Hepa-1c1c7 cells were used to study the cell growth inhibition capacity of these fractions by MTT assay. Their capacity of altering cell cycle and possible induction of apoptosis was examined using FACS analysis. RESULTS: The original polyphenolic fraction OW, which contained gallic acid (GA), (+)-catechin (Cat), (-)-epicatechin (Ec), glycosylated flavonols (F) and procyanidin oligomers was fractionated into fraction I, composed of monomers and small oligomers, and fraction II that included flavonols and procyanidin oligomers of higher molecular weight. The three polyphenolic fractions tested showed quite similar antiradical activity, although fraction I was the most potent antiradical agent (lowest ED(50) value: 9 microg). Fraction II was the least potent cell growth inhibitor (highest IC(50) value: 100 microg/ml) but showed the strongest effect on the cell cycle of Hepa-1c1c7, inducing apoptosis in those cells. The original fraction OW was demonstrated to have the most potent cell growth inhibition effect (lowest IC(50) value: 43 microg/ml). However, it only appeared to alter cell cycle of Hepa-1c1c7 at concentrations higher than its IC(50) and did not induce apoptosis in those cells. A similar effect on cell cycle and apoptosis was encountered for fraction I. CONCLUSIONS: The polyphenolic fractions tested in this study were potent antiradical agents and exerted an antiproliferative effect in mouse hepatoma Hepa-1c1c7 cells; the fraction with the highest degree of polimerization and galloylation (fraction II) had the most influence on the cell cycle and induction of apoptosis on Hepa-1c1c7.

Animals↗
PubMed2003

Mathematical modelling of the urea cycle. A numerical investigation into substrate channelling.

Metabolite channelling, the process in which consecutive enzymes have confined substrate transfer in metabolic pathways, has been proposed as a biochemical mechanism that has evolved because it enhances catalytic rates and protects unstable intermediates. Results from experiments on the synthesis of radioactive urea [Cheung, C., Cohen, N.S. & Raijman, L (1989) J. Biol. Chem.264, 4038-4044] have been interpreted as implying channelling of arginine between argininosuccinate lyase and arginase in permeabilized hepatocytes. To investigate this interpretation further, a mathematical model of the urea cycle was written, using Mathematica it simulates time courses of the reactions. The model includes all relevant intermediates, peripheral metabolites, and subcellular compartmentalization. Analysis of the output from the simulations supports the argument for a high degree of, but not absolute, channelling and offers insights for future experiments that could shed more light on the quantitative aspects of this phenomenon in the urea cycle and other pathways.

Arginase↗
PubMed2002

Recent advances in the study of epigenetic effects induced by the phycotoxin okadaic acid.

Okadaic acid (OA) is a phycotoxin produced by dinoflagellates. It accumulates in the digestive tracts of shellfish causing diarrhetic shellfish poisoning (DSP) in consumers. OA is a tumour promoter, and an inhibitor of both protein phosphatases and protein synthesis. OA induces DNA adducts, suggesting it may be carcinogenic. Since the Ames test without S(9) was negative, but a mutagenesis test was positive in mammalian cells, the question as to whether its molecular mechanism is genotoxic or epigenetic became unavoidable. Therefore, experiments were performed to search for epigenetic effects, since evidence for DNA-adduct formation using the gamma-(32)P-ATP post-labelling method was not obtained. We found that OA is a potent inducer of lipid peroxidation in human intestinal cells (Caco-2) at low concentrations (0.75-7.5 ng/ml versus IC50 of 15 ng/ml) with increased rates of 8-OH-dG and m(5)dC formation causing CG to AT transversion mutations and gene deregulation, respectively. The transcription and translation of connexin 43-specific mRNA were inhibited, and 3H-uridine incorporation in RNA was concomitantly increased. Consequently gap junction intracellular communication (GJIC) was inhibited, making possible cellular anarchic proliferation. Higher OA concentrations also disorganized the cellular cytoskeleton, since both actin and tubulin formations were impaired. Our results suggest that OA may induce tumours via an epigenetic mechanism.

Caco-2 Cells↗
PubMed2002

Valorization of grape (Vitis vinifera) byproducts. Antioxidant and biological properties of polyphenolic fractions differing in procyanidin composition and flavonol content.

Many byproducts and wastes generated by agroindustries contain polyphenols with potential application as food antioxidants and preventive agents against skin cancer and other diseases. The performance of polyphenolic fractions from Parellada grape (Vitis vinifera) pomace as antioxidants in different physicochemical environments was tested. Fractions containing oligomers with mean degree of polymerization between 3 and 4 and percentage galloylation ca. 30% were the most potent free radical scavengers and efficient antioxidants in an oil-in-water emulsion. A fraction including glycosylated flavonols was also efficient in the emulsion. All the fractions showed low aquatic toxicity and weak influence on proliferation of human melanoma cells.

Antioxidants↗
PubMed2002

Fermented wheat germ extract inhibits glycolysis/pentose cycle enzymes and induces apoptosis through poly(ADP-ribose) polymerase activation in Jurkat T-cell leukemia tumor cells.

The fermented extract of wheat germ, trade name Avemar, is a complex mixture of biologically active molecules with potent anti-metastatic activities in various human malignancies. Here we report the effect of Avemar on Jurkat leukemia cell viability, proliferation, cell cycle distribution, apoptosis, and the activity of key glycolytic/pentose cycle enzymes that control carbon flow for nucleic acid synthesis. The cytotoxic IC(50) concentration of Avemar for Jurkat tumor cells is 0.2 mg/ml, and increasing doses of the crude powder inhibit Jurkat cell proliferation in a dose-dependent fashion. At concentrations higher than 0.2 mg/ml, Avemar inhibits cell growth by more than 50% (72 h of incubation), which is preceded by the appearance of a sub-G(1) peak on flow histograms at 48 h. Laser scanning cytometry of propidium iodide- and annexin V-stained cells indicated that the growth-inhibiting effect of Avemar was consistent with a strong induction of apoptosis. Inhibition by benzyloxycarbonyl-Val-Ala-Asp fluoromethyl ketone of apoptosis but increased proteolysis of poly(ADP-ribose) indicate caspases mediate the cellular effects of Avemar. Activities of glucose-6-phosphate dehydrogenase and transketolase were inhibited in a dose-dependent fashion, which correlated with decreased (13)C incorporation and pentose cycle substrate flow into RNA ribose. This decrease in pentose cycle enzyme activities and carbon flow toward nucleic acid precursor synthesis provide the mechanistic understanding of the cell growth-controlling and apoptosis-inducing effects of fermented wheat germ. Avemar exhibits about a 50-fold higher IC(50) (10.02 mg/ml) for peripheral blood lymphocytes to induce a biological response, which provides the broad therapeutic window for this supplemental cancer treatment modality with no toxic effects.

Apoptosis↗
PubMed2002

Dependence of control coefficient distribution on the boundaries of a metabolic system: a generalized analysis of the effects of additional input and output reactions to a linear pathway.

Both experimental and theoretical studies of metabolism are likely to relate to a segment that has been isolated for analytical purposes. In practice, it will be embedded in the whole of cellular metabolism. Thus, it is necessary to consider how conclusions about the control of an isolated pathway may be modified in this wider context where the input and output metabolites are considered as variables of cellular metabolism. Here, we analyse the effect of expanding a linear metabolic pathway by adding an extra input or an extra output. In particular, we analyse the effect of the elasticities of the extra steps on control coefficients. We derive matrix algebraic relationships for obtaining flux and concentration control coefficients from expressions depending on these extra elasticities and on parameters (elasticities and control coefficients) of the original pathway. These equations can be shown in certain cases to be generalized versions of earlier rescaling relationships and to be related to top-down analysis, but also apply where the new variable metabolite of the expanded pathway is an effector of more than one step of the original pathway. We use our relationships to analyse the dependence or independence of control coefficients upon these extra elasticities for the published analyses of the pathway of mammalian serine biosynthesis (Fell & Snell, 1988) and Escherischia coli threonine biosynthesis (Chassagnole et al., 2001). The same analysis can be applied to determine whether the transport reactions of substrates and products of a pathway in and out of a cell need to be included in estimations of the control coefficients of the enzymes.

Animals↗
PubMed2002

Metabolic profiling of cell growth and death in cancer: applications in drug discovery.

Metabolic profiling using stable-isotope tracer technology enables the measurement of substrate redistribution within major metabolic pathways in living cells. This technique has demonstrated that transformed human cells exhibit profound metabolic shifts and that some anti-cancer drugs produce their effects by forcing the reversion of these metabolic changes. By revealing tumor-specific metabolic shifts in tumor cells, metabolic profiling enables drug developers to identify the metabolic steps that control cell proliferation, thus aiding the identification of new anti-cancer targets and screening of lead compounds for anti-proliferative metabolic effects.

Antineoplastic Agents↗
PubMed2002

Product dependence and bifunctionality compromise the ultrasensitivity of signal transduction cascades.

Covalent modification cycles are ubiquitous. Theoretical studies have suggested that they serve to increase sensitivity. However, this suggestion has not been corroborated experimentally in vivo. Here, we demonstrate that the assumptions of the theoretical studies, i.e., irreversibility and absence of product inhibition, were not trivial: when the conversion reactions are close to equilibrium or saturated by their product, "zero-order" ultrasensitivity disappears. For high sensitivities to arise, not only substrate saturation (zero-order) but also high equilibrium constants and low product saturation are required. Many covalent modification cycles are catalyzed by one bifunctional 'ambiguous' enzyme rather than by two independent proteins. This makes high substrate concentration and low product concentration for both reactions of the cycle inconsistent; such modification cycles cannot have high responses. Defining signal strength as ratios of modified (e.g., phosphorylated) over unmodified protein, signal-to-signal response sensitivity equals 1: signal strength should remain constant along a cascade of ambiguous modification cycles. We also show that the total concentration of a signalling effector protein cannot affect the signal emanating from a modification cycle catalyzed by an ambiguous enzyme if the ratio of the two forms of the effector protein is not altered. This finding may explain the experimental result that the pivotal signal transduction protein PII plus its paralogue GlnK do not control steady-state N-signal transduction in Escherichia coli. It also rationalizes the absence of strong phenotypes for many signal-transduction proteins. Emphasis on extent of modification of these proteins is perhaps more urgent than transcriptome analysis.

Enzymes↗
PubMed2002

Metabolic control analysis aimed at the ribose synthesis pathways of tumor cells: a new strategy for antitumor drug development.

Metabolic control analysis predicts that effects on tumor growth are likely to be obtained with lower concentrations of drug, if an enzyme with a high control coefficient on tumor growth is being inhibited. Here we measure glucose-6-phosphate dehydrogenase (G6PDH) control coefficient on in vivo tumor growth using mice bearing Ehrlich ascites tumor cells. We used dehydroepiandrosterone-sulphate (DHEA-S), an uncompetitive inhibitor of this enzyme and the in situ cytochemical method to measure the enzyme activity changes that accompany changes on tumor cell growth. This method ensures that the enzyme activity determined is the one existing in the in situ conditions and enables computing a control coefficient in in situ conditions. From the data obtained on tumor cell number and the in situ enzyme activities in absence and presence of DHEA-S, a control coefficient of 0.41 for G6PDH on tumor cell growth was computed. This value is approximately the half of the transketolase control coefficient value of 0.9 previously reported. Moreover, the use of in situ methods to assess enzyme activities, applied for first time for the calculation of control coefficients in this study, opens new avenues to the use of uncompetitive inhibitors for the measurement of in situ control coefficients.

Animals↗
PubMed2002

Sensitivity analysis of metabolic cascades catalyzed by bifunctional enzymes.

Covalent modification/demodification cycles are common in metabolism. When the modification and demodification steps are carried out by two independent enzymes, the degree of modification can be ultrasensitive to the total concentration of either catalyst. We recently showed that the degree of modification of a target molecule cannot exhibit ultrasensitivity to the free concentrations of effectors that decide whether a bifunctional enzyme acts as modifier or demodifier. However, here we can now demonstrate that the degree of modification of a target molecule can display ultrasensitivity to the total, rather than free, concentrations of such effectors. Our results clarify some general aspects of ultrasensitive responses to effectors, including competitive inhibitors, in mono-cyclic cascades.

Catalysis↗
PubMed2002

Modulation of metabolite concentrations with no net effect on fluxes.

The concentration of a metabolite in a metabolic system can be varied without affecting any other concentrations or any fluxes by varying the concentrations of two inhibitors, one a competitive inhibitor of the enzyme that produces the metabolite, the other a competitive inhibitor of the enzyme that consumes it. The two concentrations need to be varied in opposite directions in such a way that the they add up to 100% when each is expressed as a percentage of the concentration that gives the desired flux in the absence of the other. The general approach can be extended to systems in which the inhibited enzymes do not catalyse consecutive reactions.

Enzyme Inhibitors↗