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M Cascante

Publications and source records attributed to M Cascante.

At least 37 records · Page 2Linked to original sources
PubMed2000

Organization-dependent effects of toxic bivalent ions microtubule assembly and glycolysis.

The effects of bivalent ions on tubulin dynamics and the upper phase of glycolysis were investigated at different organization levels in vitro. Cu2+, Cd2+, Hg2+ and CrO4(2-) inhibit the tubulin polymerization at an IC50 of 14-24 microM with high cooperativity and also induce microtubule disassembly. The apparent binding constants of the ions to tubulin, estimated by fluorescence quenching, vary between 6 and 28 microM. BIAcore measurements for tubulin-tubulin interaction suggest that the presence of Cu2+ affects neither koff nor kon, but the amount of the bound tubulin. While the inhibitory effect of Cu2+ on tubulin polymerization is partially abolished by cross-linking of microtubules with substoichiometric amounts of phosphofructokinase or decoration of tubules with cytosolic proteins, in the presence of kinase but not with cytosolic proteins the tubules are resistant to CrO4(2-). No inhibitory effect of Cu2+ or CrO4(2-) on microtubule assembly was detected in the MAP-containing cytosolic fraction. Electron microscopy revealed that tubules assembled in the presence of Cu2+ or CrO4(2-) ions contain aggregates of thread-like oligomers that are less conspicuous in the presence of cytosolic proteins. Cu2+, Cd2+, and Hg2+ inhibit the glycolytic flux in the cytosolic fraction characterized at equilibrium by an IC50 of 10-14 microM with high cooperativity. Tubulin diminishes the inhibitory effect of the cations. These data indicate that the responses elicited by the bivalent ions are highly dependent on the supramolecular organization of the systems.

Acetates↗
PubMed2000

Role of thiamin (vitamin B-1) and transketolase in tumor cell proliferation.

Metabolic control analysis predicts that stimulators of transketolase enzyme synthesis such as thiamin (vitamin B-1) support a high rate of nucleic acid ribose synthesis necessary for tumor cell survival, chemotherapy resistance, and proliferation. Metabolic control analysis also predicts that transketolase inhibitor drugs will have the opposite effect on tumor cells. This may have important implications in the nutrition and future treatment of patients with cancer.

Cell Division↗
PubMed2000

[Primary retinal vasculitis].

CASE REPORT: We present the case of a 22-year-old Caucasian female who consulted because of a brief episode of blurred vision in her right eye. Funduscopic findings in right eye included marked venous tortuousness and roundish paramacular haemorrhages. A slight venous tortuousness in left eye. Fluorescein angiography showed a segmentary retinal vasculitis affecting medium calibre vessels of the posterior pole. Complementary examinations ruled out any systemic involvement. After therapy with oral corticoesteroids the case evolves to remission. DISCUSSION: The diagnosis of retinal vasculitis is achieved by the performance of ophthalmoscopy and fluorescein angiography. Prior studies have ruled out the need for complementary examinations to find any systemic involvement in patients.

Adult↗
PubMed1999

Combined enhancement of microtubule assembly and glucose metabolism in neuronal systems in vitro: decreased sensitivity to copper toxicity.

Brain cell-free extract greatly stimulates the polymerization rate of purified tubulin with a reduction of the nucleation period and without a significant alteration of the final assembly state. This effect is mimicked by neuroblastoma extract at 10-fold lower extract concentration, but not by excess muscle extract. Copper inhibits microtubule assembly in vitro but in the presence of brain extract the copper effect is suspended. Electron microscopic images showed that intact microtubules are formed and decorated by cytosolic proteins in the absence and presence of copper, while the copper alone induces the formation of S-shaped sheets and oligomeric threads. The flux of triosephosphate formation from glucose is enhanced by microtubules in brain extract, but not in muscle extract. Copper inhibits the glycolytic flux; however, the presence of microtubules not only suspends the inhibition by copper but the activation of glycolysis by microtubules is also preserved. We conclude that the organization of neuronal proteins modifies both the rates of microtubule assembly and glycolysis, and reduces their sensitivities against the inhibition caused by copper.

Animals↗
PubMed1999

Oxythiamine and dehydroepiandrosterone induce a G1 phase cycle arrest in Ehrlich's tumor cells through inhibition of the pentose cycle.

Transketolase (TK) reactions play a crucial role in tumor cell nucleic acid ribose synthesis utilizing glucose carbons, yet, current cancer treatments do not target this central pathway. Experimentally, a dramatic decrease in tumor cell proliferation after the administration of the TK inhibitor oxythiamine (OT) was observed in several in vitro and in vivo tumor models. Here, we demonstrate that pentose cycle (PC) inhibitors, OT and dehydroepiandrosterone (DHEA), efficiently regulate the cell cycle and tumor proliferation processes. Increasing doses of OT or DHEA were administered by daily intraperitoneal injections to Ehrlich's ascites tumor hosting mice for 4 days. The tumor cell number and their cycle phase distribution profile were determined by DNA flow histograms. Tumors showed a dose dependent increase in their G0-G1 cell populations after both OT and DHEA treatment and a simultaneous decrease in cells advancing to the S and G2-M cell cycle phases. This effect of PC inhibitors was significant, OT was more effective than DHEA, both drugs acted synergistically in combination and no signs of direct cell or host toxicity were observed. Direct inhibition of PC reactions causes a G1 cell cycle arrest similar to that of 2-deoxyglucose treatment. However, no interference with cell energy production and cell toxicity is observed. PC inhibitors, specifically ones targeting TK, introduce a new target site for the development of future cancer therapies to inhibit glucose utilizing pathways selectively for nucleic acid production.

Animals↗
PubMed1999

Application of metabolic control analysis to the study of toxic effects of copper in muscle glycolysis.

Experimental and model studies have been performed to characterise the effects of Cu2+ on the activities of individual glycolytic enzymes and on the flux and internal metabolite concentrations of the upper part of glycolysis in mouse muscle extracts. Cu2+ significantly inhibited the triosephosphate production from glucose with an IC50 of about 6.0 microM. At a similar extension Cu2+ inhibited hexokinase and phosphofructokinase, with an IC50 of 6.2 microM and 6.4 microM respectively, whereas the effects on the activities of aldolase, phosphoglucose isomerase and the internal metabolite levels were not significant. Flux control coefficients and flux response coefficients were determined in the presence of copper concentrations between 0 and 10 microM. The same values of flux control coefficients for hexokinase and for phosphofructokinase (0.8 and 0.2 respectively) were found in absence and in presence of copper. At Cu2+ equal to the flux IC50, the response coefficient was -1. The elasticity coefficients for hexokinase and phosphofructokinase at Cu2+ equal to the IC50 were also -1. A mathematical model was used to analyze the effect of copper on glycolysis under different conditions using experimental kinetic parameters and rate equations for enzymatic reactions of the upper part of glycolysis.

Animals↗
PubMed1999

Cytotoxicity and genotoxicity of capsaicin in human neuroblastoma cells SHSY-5Y.

Capsaicin, a natural product of Capsicum species, induces excitation of pain perception at nociceptive terminals. Our previous studies have shown that capsaicin inhibits protein synthesis in cultured monkey kidneys cells (Vero cells) and in primoculture of rat astrocytes. We have now investigated the effect of capsaicin on human neuroblastoma cells SHSY-5Y. The cytotoxicity has been assessed by incorporation of [(3)H]L-leucine into cellular protein in the presence of capsaicin and the genotoxicity has been evaluated using the comet assay and the fragmentation assay after incubation of neuroblastoma cells with 25-100 microM capsaicin. The concentration required to inhibit 50% of the protein synthesis (IC(50)) was found to be 60 microM after incubation with the toxin during one cellular cycle (5 days) of SHSY-5Y. The results of the comet test and DNA fragmentation assay clearly suggest that capsaicin is able to induce DNA strand breaks already with concentrations in the range of 50 microM, corresponding to 29.3 microM of capsaicin not bound to alpha-1 acid glycoprotein. Several daily topical applications of preparations containing 0.075% of capsaicin could lead to blood capsaicin concentration of this order of magnitude following transdermal passage (5% of the total quantity applied). Because DNA strand breaks or DNA lesions may affect cellular functions, lead to cell death and/or mutagenesis, our data in case of inappropriate DNA repair may have important implications for the possible health threats of capsaicin, specially in the case of misuse of capsaicin preparations in pathological situations.

Capsaicin↗
PubMed1999

New semisynthetic vinca alkaloids: chemical, biochemical and cellular studies.

A new semisynthetic anti-tumour bis-indol compound, KAR-2 [3'-(beta-chloroethyl)-2',4'-dioxo-3,5'-spiro-oxazolidino-4-dea cetoxy-vinblastine] with lower toxicity than vinca alkaloids used in chemotherapy binds to calmodulin but, in contrast to vinblastine, does not exhibit anti-calmodulin activity. To investigate whether the modest chemical modification of bis-indol structure is responsible for the lack of anti-calmodulin potency and for the different pharmacological effects, new derivatives have been synthesized for comparative studies. The synthesis of the KAR derivatives are presented. The comparative studies showed that the spiro-oxazolidino ring and the substitution of a formyl group to a methyl one were responsible for the lack of anti-calmodulin activities. The new derivatives, similar to the mother compounds, inhibited the tubulin assembly in polymerization tests in vitro, however their inhibitory effect was highly dependent on the organization state of microtubules; bundled microtubules appeared to be resistant against the drugs. The maximal cytotoxic activities of KAR derivatives in in vivo mice hosting leukaemia P388 or Ehrlich ascites tumour cells appeared similar to that of vinblastine or vincristine, however significant prolongation of life span could be reached with KAR derivatives only after the administration of a single dose. These studies plus data obtained using a cultured human neuroblastoma cell line showed that KAR compounds displayed their cytotoxic activities at significantly higher concentrations than the mother compounds, although their antimicrotubular activities were similar in vitro. These data suggest that vinblastine/vincristine damage additional crucial cell functions, one of which could be related to calmodulin-mediated processes.

Animals↗
PubMed1999

Advantages and disadvantages of aggregating fluxes into synthetic and degradative fluxes when modelling metabolic pathways.

It is now widely accepted that mathematical models are needed to predict the behaviour of complex metabolic networks in the cell, in order to have a rational basis for planning metabolic engineering with biotechnological or therapeutical purposes. The great complexity of metabolic networks makes it crucial to simplify them for analysis, but without violating key principles of stoichiometry or thermodynamics. We show here, however, that models for branched complex systems are sometimes obtained that violate the stoichiometry of fluxes at branch points and as a result give unrealistic metabolite concentrations at the steady state. This problem is especially important when models are constructed with the S-system form of biochemical systems theory. However, the same violation of stoichiometry can occur in metabolic control analysis if control coefficients are assumed to be constant when trying to predict the effects of large changes. We derive the appropriate matrix equations to analyse this type of problem systematically and to assess its extent in any given model.

Kinetics↗
PubMed1998

Metabolic design: how to engineer a living cell to desired metabolite concentrations and fluxes.

A biotechnological aim of genetic engineering is to increase the intracellular concentration or secretion of valuable compounds, while making the other concentrations and fluxes optimal for viability and productivity. Efforts to accomplish this based on over-expression of the enzyme, catalyzing the so-called "rate-limiting step," have not been successful. Here we develop a method to determine the enzyme concentrations that are required to achieve such an aim. This method is called Metabolic Design Analysis and is based on the perturbation method and the modular ("top-down") approach-formalisms that were first developed for the analysis of biochemical regulation such as, Metabolic Control Analysis. Contrary to earlier methods, the desired alterations of cellular metabolism need not be small or confined to a single metabolite or flux. The limits to the alterations of fluxes and metabolite concentrations are identified. To employ Metabolic Design Analysis, only limited kinetic information concerning the pathway enzymes is needed.

Biotechnology↗
PubMed1998

Control analysis of metabolic systems involving quasi-equilibrium reactions.

Reactions for which the rates are extremely sensitive to changes in the concentrations of variable metabolite concentrations contribute little to the control of biochemical reaction networks. Yet they do interfere with the calculation of the system's behaviour, both in terms of numerical integration of the rate equations and in terms of the analysis of metabolic control. We here present a way to solve this problem systematically for systems with time hierarchies. We identify the fast reactions and fast metabolites, group them apart from the other ("slow") reactions and metabolites, and then apply the appropriate quasi-equilibrium condition for the fast subsystem. This then makes it possible to eliminate the fast reactions and their elasticity coefficients from the calculations, allowing the calculation of the control coefficients of the slow reactions in terms of the elasticity coefficients of the slow reactions. As expected, the elasticity coefficients of the fast reactions drop out of the calculations, and they are irrelevant for control at the time resolution of the steady state of the slow reactions. The analysis, when applied iteratively, is expected to be particularly valuable for the control analysis of living cells, where a time hierarchy exists, the fastest being at the level of enzyme kinetics and the slowest at gene expression.

Cells↗
PubMed1998

Analysis of abnormalities in purine metabolism leading to gout and to neurological dysfunctions in man.

A modelling approach is used to analyse diseases associated with purine metabolism in man. The specific focus is on deficiencies in two enzymes, hypoxanthine:guanine phosphoribosyltransferase and adenylosuccinate lyase. These deficiencies can lead to a number of symptoms, including neurological dysfunctions and mental retardation. Although the biochemical mechanisms of dysfunctions associated with adenylosuccinate lyase deficiency are not completely understood, there is at least general agreement in the literature about possible causes. Simulations with our model confirm that accumulation of the two substrates of the enzyme can lead to significant biochemical imbalance. In hypoxanthine:guanine phosphoribosyltransferase deficiency the biochemical mechanisms associated with neurological dysfunctions are less clear. Model analyses support some old hypotheses but also suggest new indicators for possible causes of neurological dysfunctions associated with this deficiency. Hypoxanthine:guanine phosphoribosyltransferase deficiency is known to cause hyperuricaemia and gout. We compare the relative importance of this deficiency with other known causes of gout in humans. The analysis suggests that defects in the excretion of uric acid are more consequential than defects in uric acid synthesis such as hypoxanthine:guanine phosphoribosyltransferase deficiency.

AMP Deaminase↗
PubMed1998

Physical constraints in the synthesis of glycogen that influence its structural homogeneity: a two-dimensional approach.

Several aspects of glycogen optimization as an efficient fuel storage molecule have been studied in previous works: the chain length and the branching degree. These results demonstrated that the values of these variables in the cellular molecule are those that optimize the structure-function relationship. In the present work we show that structural homogeneity of the glycogen molecule is also an optimized variable that plays an important role in its metabolic function. This problem was studied by means of a two-dimensional approach, which allowed us to simplify the very complicated structure of glycogen. Our results demonstrate that there is a molecular size limit that guarantees the structural homogeneity, beyond which the structure of the molecule degenerates, as many chains do not grow. This strongly suggests that such a size limit is precisely what the molecule possesses in the cell.

Biophysical Phenomena↗
PubMed1998

Mathematical models of purine metabolism in man.

Experimental and clinical data on purine metabolism are collated and analyzed with three mathematical models. The first model is the result of an attempt to construct a traditional kinetic model based on Michaelis-Menten rate laws. This attempt is only partially successful, since kinetic information, while extensive, is not complete, and since qualitative information is difficult to incorporate into this type of model. The data gaps necessitate the complementation of the Michaelis-Menten model with other functional forms that can incorporate different types of data. The most convenient and established representations for this purpose are rate laws formulated as power-law functions, and these are used to construct a Complemented Michaelis-Menten (CMM) model. The other two models are pure power-law-representations, one in the form of a Generalized Mass Action (GMA) system, and the other one in the form of an S-system. The first part of the paper contains a compendium of experimental data necessary for any model of purine metabolism. This is followed by the formulation of the three models and a comparative analysis. For physiological and moderately pathological perturbations in metabolites or enzymes, the results of the three models are very similar and consistent with clinical findings. This is an encouraging result since the three models have different structures and data requirements and are based on different mathematical assumptions. Significant enzyme deficiencies are not so well modeled by the S-system model. The CMM model captures the dynamics better, but judging by comparisons with clinical observations, the best model in this case is the GMA model. The model results are discussed in some detail, along with advantages and disadvantages of each modeling strategy.

Animals↗
PubMed1998

Effects of feedback inhibition on transit time in a linear pathway of Michaelis-Menten-type reactions.

An analysis of the effects of external and internal metabolites on the steady-state behavior of linear pathways comprising a sequence of three Michaelis-Menten-type reactions with and without a simple feedback inhibition (i.e. an interaction of an internal metabolite with the pathway) is performed with respect to the transit time tau by its formulation as rectangular-hyperbolic functions of the flux J, instead of direct expressions in terms of the external metabolite concentrations. For a given concentration of the external metabolite M1 (substrate of the pathway) or M4 (product of the pathway), the flux J has a lower value in the pathway with feedback inhibition than in the pathway without feedback inhibition. With variation in the M1 concentration the transit time tau shows a concave relationship with the flux J which is virtually identical for both pathways, yielding a minimum at a certain value of J. With variation in the M4 concentration the transit time tau monotonously decreases with higher value of J, and for a given value of J the feedback inhibition allows a lower transit time. This effect is enhanced with stronger feedback inhibition, and is in turn greatly reduced with higher values of total concentration and rate constants for the first enzyme in the pathway.

Enzymes↗
PubMed1998

Nonoxidative pentose phosphate pathways and their direct role in ribose synthesis in tumors: is cancer a disease of cellular glucose metabolism?

Pentose phosphate pathways (PPP) are considered important in tumor proliferation processes because of their role in supplying tumor cells with reduced NADP and carbons for intracellular anabolic processes. Direct involvement of PPP in tumor DNA/RNA synthesis is not considered as significant as in lipid and protein syntheses. Currently, PPP activity in tumor cells is measured by lactate production, which shows a moderate activity: about 4% to 7% compared with glycolysis. Recent data generated in our laboratory indicate that PPP are directly involved in ribose synthesis in pancreatic adenocarcinoma cells, through oxidative steps (< 31%) and transketolase reactions (69%). These findings raise serious questions about the adequacy of lactate in measuring PPP activity in tumors. We hypothesize that ribose, not lactate, is the major product of PPP in tumor cells. Control of both oxidative and nonoxidative PPP may be critical in the treatment of cancer. PPP are substantially involved in the proliferation of human tumors, which raises the prospect of new treatment strategies targeting specific biochemical reactions of PPP by hormones related to glucose metabolism, controlling thiamine intake, the cofactor of the nonoxidative transketolase PPP reaction, or treating cancer patients with antithiamine analogues.

Adenocarcinoma↗
PubMed1998

Inhibition of the oxidative and nonoxidative pentose phosphate pathways by somatostatin: a possible mechanism of antitumor action.

Long-acting somatostatin analogs have recently become supplemental drugs in the treatment of neurofibroma because of their marked tumor growth inhibitory effect. Somatostatin is currently under extended evaluation in other cancers as a possible supplemental drug to the treatment protocols in use. The mode of action is not known. Somatostatin has been shown to cause glucose intolerance by inhibiting glucose-6-phosphate dehydrogenase (G6PD) in fish liver. Recent data generated in our laboratory indicate that it is this pathway and the transketolase reactions of the pentose cycle (PC) which are directly involved in the ribose synthesis process of pancreatic adenocarcinoma cells. In cell culture, somatostatin alone inhibited glucose carbon recycling through the PC by 5.7%, which was increased to 19.8% in combination with oxythiamine, a competitive inhibitor of transketolase. Oxythiamine produced strong apoptosis in in-vitro hosted tumor cells. We hypothesize that somatostatin- and oxythiamine-induced antiproliferative action is mediated by the inhibition of G6PD, transketolase, or both.

Animals↗