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S Cortassa

Publications and source records attributed to S Cortassa.

At least 19 recordsLinked to original sources
PubMed2001

Why homeodynamics, not homeostasis?

Ideas of homeostasis derive from the concept of the organism as an open system. These ideas can be traced back to Heraclitus. Hopkins, Bernard, Hill, Cannon, Weiner and von Bertalanffy developed further the mechanistic basis of turnover of biological components, and Schoenheimer and Rittenberg were pioneers of experimental approaches to the problems of measuring pool sizes and dynamic fluxes. From the second half of the twentieth century, a biophysical theory mainly founded on self-organisation and Dynamic Systems Theory allowed us to approach the quantitative and qualitative analysis of the organised complexity that characterises living systems. This combination of theoretical framework and more refined experimental techniques revealed that feedback control of steady states is a mode of operation that, although providing stability, is only one of many modes and may be the exception rather than the rule. The concept of homeodynamics that we introduce here offers a radically new and all-embracing concept that departs from the classical homeostatic idea that emphasises the stability of the internal milieu toward perturbation. Indeed, biological systems are homeodynamic because of their ability to dynamically self-organise at bifurcation points of their behaviour where they lose stability. Consequently, they exhibit diverse behaviour; in addition to monotonic stationary states, living systems display complex behaviour with all its emergent characteristics, i.e., bistable switches, thresholds, waves, gradients, mutual entrainment, and periodic as well as chaotic behaviour, as evidenced in cellular phenomena such as dynamic (supra)molecular organisation and flux coordination. These processes may proceed on different spatial scales, as well as across time scales, from the very rapid processes within and between molecules in membranes to the slow time scales of evolutionary change. It is dynamic organisation under homeodynamic conditions that make possible the organised complexity of life.

Hemodynamics↗
PubMed2001

Measurement of the glycogen synthetic pathway in permeabilized cells of cyanobacteria.

A simple, rapid and reliable procedure for permeabilizing cyanobacterial cells and measuring the glycogen synthetic pathway in situ, is presented. Cells from Anabaena sp. strain PCC 7120 were permeabilized with a mixture of toluene:ethanol (1:4 v/v). Fluorescence microscopy of cells incubated with fluorescein diacetate showed Anabaena non-permeabilized cells as green fluorescents, whereas permeabilized (viable) cells exhibited the intrinsic red fluorescence. Labelled alpha-1,4-glucan was recovered when permeabilized cells were incubated with the substrates of ADP-glucose pyrophosphorylase or glycogen synthase. The kinetic and regulatory properties of both enzymes could be reproduced in situ. The simplicity of the procedure and the ability to measure in situ glucan fluxes show the methodology as useful for studying the intracellular regulation of storage polysaccharides in a photosynthetic prokaryote.

Anabaena↗
PubMed2001

Ultrasensitivity in (supra)molecularly organized and crowded environments.

The ultrasensitive response of biological systems is a more sensitive one than that expected from the classical hyperbola of Michaelis-Menten kinetics, and whose physiological relevance depends upon the range of variation of substrate or effector for which ultrasensitivity is observed. Triggering and modulation of the ultrasensitive response in enzymatic and cellular systems are reviewed. Several demonstrations of ultrasensitive behavior in cellular systems and its impact on the amplification properties in signalling cascades and metabolic pathways are also highlighted. It is shown that ubiquitous cytoskeletal proteins may up- or downmodulate ultrasensitivity under physico-chemical conditions resembling those predominant in cells.

Animals↗
PubMed2001

Involvement of nitrogen metabolism in the triggering of ethanol fermentation in aerobic chemostat cultures of Saccharomyces cerevisiae.

We have investigated whether central nitrogen metabolism may influence the triggering of ethanol fermentation in Saccharomyces cerevisiae strain CEN.PK122 grown in the presence of different N-sources (ammonia, glutamate, or glutamine) under conditions in which the carbon to nitrogen (C : N) ratio was varied. An exhaustive quantitative evaluation of yeast physiology and metabolic behavior through metabolic flux analysis (MFA) was undertaken. It is shown that ethanol fermentation is triggered at dilution rates, D (growth rate), significantly lower (D=0.070 and 0.074 h(-1) for glutamate and glutamine, respectively, and D=0.109 h(-1) for ammonia) under N- than C-limitation (approximately 0.18 h(-1) for all N-sources). A characteristic specific rate of glucose influx, q(Glc), for each N-source at Dc, i.e., just before the onset of respirofermentative metabolism, was determined (approximately 2.0, 1.5, and 2.5, for ammonia, glutamate, and glutamine, respectively). This q(Glc) was independent of the nutritional limitation though dependent on the nature of the N-source. The onset of fermentation occurs when this "threshold q(Glc)" is overcome. The saturation of respiratory activity appears not to be associated with the onset of fermentation since q(O(2)) continued to increase after Dc. It was remarkable that under respirofermentative conditions in C-limited chemostat cultures, the glucose consumed was almost completely fermented with biomass being synthesized from glutamate through gluconeogenesis. The results obtained show that the enzyme activities involved in central nitrogen metabolism do not appear to participate in the control of the overflow in carbon catabolism, which is driven toward ethanol production. The role of nitrogen metabolism in the onset of ethanol fermentation would rather be realized through its involvement in setting the anabolic fluxes directed to nitrogenous macromolecules. It seems that nitrogen-related anabolic fluxes would determine when the threshold glucose consumption rate is achieved after which ethanol fermentation is triggered.

Aerobiosis↗
PubMed2000

Chaotic dynamics and fractal space in biochemistry: simplicity underlies complexity.

In this work we attempt to analyze the coupling between the dynamics of biochemical reactions (especially chaotic dynamics), and the geometry of cytoarchitecture (especially fractal ultrastructure), because of its importance and consequences for the ultradian dynamic behaviour of cells. Fractal geometry in intracellular macromolecular assemblies suggests that chaotic dynamics occur during their organization. Non-linear interactions in and between spatial and temporal domains and over wide ranges of scales underlie the emergent properties of complex biological systems.

Biochemical Phenomena↗
PubMed2000

Dynamics of metabolism and its interactions with gene expression during sporulation in Saccharomyces cerevisiae.

The dynamics of metabolism has been shown to be involved in the triggering of events that are concurrent with sporulation of the budding yeast Saccharomyces cerevisiae. Indeed, quantitative correlations have been demonstrated between sporulation and the rate of carbon substrate or oxygen consumption, and the fluxes through gluconeogenic and glyoxylate cycle pathways. The results suggest that an imbalance between catabolic and anabolic fluxes influences the occurrence of the differentiation process. The hypothesis that the initiation of sporulation is triggered by the accumulation of an intracellular metabolite is confronted with the notion that intermediary metabolism and the expression of genes involved in sporulation interact to trigger the differentiation process. Several pieces of evidence indicate that derepression of the gluconeogenic pathway is crucial for the initiation of sporulation. One of the possible pathways through which glucose repression hampers sporulation might be the repression of gluconeogenesis as well as that of respiratory activity, in turn modulating the expression of IMEL++. The stages defined in the dynamics of sporulating cultures, namely readiness and commitment, are related to metabolic events associated with sporulation. An interpretation in terms of metabolic flux dynamics is given to the reversal of commitment occurring when the normal progression to sporulation is somehow blocked. The quantitative data are here integrated in a model attempting to simulate the dynamics of metabolic as well as cellular events during sporulation. The model is envisaged as a test of the hypothesis that an imbalance between anabolism and catabolism is involved in initiation of the sporulation process. It is proposed that such an imbalance may be a signal for differential gene expression associated with the differentiation pathway.

Cell Differentiation↗
PubMed2000

Effects of stress on cellular infrastructure and metabolic organization in plant cells

Ample evidence shows the role of cytoskeleton mainly in cell division, cell form, and general orientation by the perception of physical forces such as gravity and mechanical ones in plant cells. However, the problem of how cytoskeleton organization and its dynamics at the cellular level in turn affects main metabolic pathways of gene expression and cellular energetics is yet unsolved. The response given by cells to environmental challenges such as stress responses is crucially dependent on the organization of their architecture. Drought, high salinity, and low temperature are sensed by plants as a water stress condition. The latter is known to entrain a series of physiological and metabolic changes at the cellular level. This review hypothesizes that the cytoskeletal network of plant cells and tissues may transduce environmental stress into changes in the organization and dynamics of metabolism and gene expression. Accordingly, experimental evidence concerning the current models of cytoplasmic architecture that have emerged in recent years and the effects of stress on the cytostructure are analyzed.

Journal Article↗
PubMed1999

Quantitation of the effects of disruption of catabolite (de)repression genes on the cell cycle behavior of Saccharomyces cerevisiae.

We have studied the effect of disrupting catabolite (de)repression genes SNF1, SNF4, and MIG1 on the cell cycle behavior of the CEN. PK122 wild type (WT) strain of Saccharomyces cerevisiae by flow cytometry in glucose-limited chemostat cultures or batch growth in the presence of different carbon sources. Through a combination of flow cytometry of propidium iodide-stained cells and mathematical modeling we showed that the deletion of the SNF4 gene provoked a decrease in the length of G1 with respect to the WT strain along with a smaller difference in the cell cycle length of parent and daughter cells. snf1 and mig1 mutants exhibited slightly shorter G1 respect to the WT. Additionally, in the mig1 mutant the cell cycle length of parent and daughter cells was slightly altered. The results obtained are in agreement with the view that the SNF4 gene is involved in the regulation of cell cycle in yeast.

Cell Cycle↗
PubMed1998

Catabolite repression mutants of Saccharomyces cerevisiae show altered fermentative metabolism as well as cell cycle behavior in glucose-limited chemostat cultures.

In glucose-limited continuous cultures, a Crabtree positive yeast such as Saccharomyces cerevisiae displays respiratory metabolism at low dilution rates (D) and respiro-fermentative metabolism at high D. We have studied the onset of ethanol production and cell cycle behavior in glucose-limited chemostat cultures of the wild type S. cerevisiae strain CEN.PK122 (WT) and isogenic mutants, snf1 (cat1) and snf4 (cat3) defective in proteins involved in catabolite derepression and the mutant in glucose repression mig1 (cat4). The triggering of fermentative metabolism was dependent upon catabolite repression properties of yeast and was coincident with a significant decrease of G1 length. WT cells of the strain CEN.PK122 displayed respiratory metabolism up to a D of 0.2 h-1 and exhibited longer G1 lengths than the snf1 and snf4 mutants that started fermenting after a D of 0.1 and 0.15 h-1, respectively. The catabolite derepression mutant snf4 showed a significant decrease in the duration of G1 with respect to the WT. An increase of 300% to 400% in the expression of CDC28 (CDC28-lacZ) with a noticeable shortening in G1 to values lower than approximately 150 min, was detected in the transformed wild type CEN.SC13-9B in glucose-limited chemostat cultures. The expression of CDC28-lacZ was analyzed in the wild type and isogenic mutant strains growing at maximal rate on glucose or in the presence of ethanol or glycerol. Two- to three-fold lower expression of the CDC28-lacZ fusion gene was detected in the snf1 or snf4 disruptants with respect to the WT and mig1 strains in the presence of all carbon sources. This effect was further shown to be growth rate-dependent exhibiting apparently, a threshold effect in the expression of the fusion gene with respect to the length of G1, similar to that shown in chemostat cultures. At the onset of fermentation, the control of the glycolytic flux was highly distributed between the uptake, hexokinase, and phosphofructokinase steps. Particularly interesting was the fact that the snf1 mutant exhibited the lowest fluxes of ethanol production, the highest of respiration and correspondingly, the branch to the tricarboxylic acid cycle was significantly rate-controling of glycolysis.

Biotechnology↗
PubMed1997

Fluorescent measurement of the intracellular pH during sporulation of Saccharomyces cerevisiae.

This work reports the intracellular pH (pHi) dynamics of Saccharomyces cerevisiae cells in sporulation medium. Cells loaded with the pH-sensitive dye carboxy-seminaphthorhodafluor-1 (C.SNARF-1) exhibited an alkalization of the pHi following the extracellular pH during sporulation in the absence of buffer and almost no change in pHi or delta pH when sporulation was carried out in buffered medium. The results indicate that the pH gradient does not appear to be directly involved in the regulation of acetate uptake during sporulation. However, the alkalization of pHi by eliciting a decrease in metabolic fluxes could account for a lower demand for acetate.

Benzopyrans↗
PubMed1997

Modulation of sporulation and metabolic fluxes in Saccharomyces cerevisiae by 2 deoxy glucose.

Quantitative studies of metabolic fluxes during Saccharomyces cerevisiae sporulation on acetate in the presence of the glucose analog, 2-deoxy glucose (2dG) are reported. We have studied the inhibition of sporulation and associated catabolic or anabolic fluxes by 2dG. Sporulation frequencies decreased from 50% to 2% asci per cell at 2dG concentrations in the range of 0.03 to 0.30 g l-1, respectively. Under the same conditions, the acetate consumption flux was inhibited up to 60% and the glyoxylate cycle and gluconeogenic fluxes decreased from 0.7 and 0.3 mmol h-1 g-1 dw, respectively, to negligible values. We observed a linear correlation of the acetate consumption rate with the sporulation frequency by varying the 2dG concentration. The linear correlation was also verified between the frequency of sporulation and the fluxes through glyoxylate cycle and gluconeogenic pathways. In addition, the same association of inhibition of sporulation and metabolic fluxes was found in other S. cerevisiae strains displaying different potentials of sporulation. The results presented suggest that inhibition of sporulation in the presence of the glucose analog may be attributed, at least in part, to the inhibition of anabolic fluxes and might be associated with catabolite repression.

Acetates↗
PubMed1996

Metabolic fluxes regulate the success of sporulation in Saccharomyces cerevisiae.

In this work we investigated to what extent cellular metabolism and energetics regulate sporulation in Saccharomyces cerevisiae and which metabolic pathways are involved in such regulation. Sporulation, meiosis, and associated metabolic fluxes in S. cerevisiae strain CH1211 were studied in several experimental protocols involving changes of carbon source (acetate, lactate, or pyruvate) or cell density in sporulation medium, or changing the phase of batch growth at which cells were harvested before transfer to sporulation medium. In acetate-based sporulation medium, the rate at which cells utilized glyoxylate and gluconeogenic pathways correlated positively with the percentage of asci per cell at 72 h. In contrast, in lactate sporulation medium the frequency of sporulation correlated negatively with both the rate of lactate consumption and the fluxes through gluconeogenesis and the pyruvate-carboxylase catalyzed step. In the presence of lactate, the respiratory capacity did correlate positively with the percentage of asci per cell. The experimental data suggest that acetate limits fluxes to anabolic precursors during sporulation. In contrast, sporulation on lactate appears to be influenced by catabolic processes or, even more precisely, by the respiratory capacity of yeast cells. The results obtained are discussed in terms of the hypothesis that an imbalance between anabolic and catabolic fluxes may be required for an efficient sporulation.

Acetates↗
PubMed1996

Heterogeneous distribution and organization of cytoskeletal proteins drive differential modulation of metabolic fluxes.

On the basis of experimental data obtained in vitro, we propose that differential segregation of actin and tubulin in the cytoplasm may be a regulatory mechanism of metabolic fluxes. The results presented point out that the same enzymes may be differentially modulated at different locations in the cytoplasm, depending on the cytoskeletal protein present at that location, its concentration, polymeric status, or geometric arrangement. Essentially, actin or microtubular protein would exert their effect on enzymatic catalysis through displacement of the equilibrium of enzyme oligomers either to active or less active species. The latter was corroborated by mathematical modeling of the dynamic coupling between microtubular protein assembly-disassembly and pyruvate kinase activity. From these results, a precise biochemical meaning can be given to the putative linkage existing between the mechanisms by which cells rearrange their cytoplasmic architecture and the dynamics of biochemical reactions taking place concomitantly.

Actins↗
PubMed1996

Metabolic rates during sporulation of Saccharomyces cerevisiae on acetate.

We have quantified yeast carbon and oxygen consumption fluxes and estimated anabolic fluxes through glyoxylate and gluconeogenic pathways under various conditions of sporulation on acetate. The percentage of sporulation reached a maximum of 55% to 60% after 48 h in sporulation medium, for cells harvested from logarithmic growth in acetate minimal medium. When cells were harvested in the stationary phase of growth before transfer to sporulation medium, the maximum percentage of sporulation decreased to 40% along with the occurrence of meiosis as could be judged by counting of bi- and tetra-nucleated cells. In both experiments, the rates of acetate and oxygen consumption decreased as a function of time when exposed to sporulation medium. Apparently, the decrease of metabolic rates was not due to alkalinization. By systematically varying the cell concentration in sporulation medium from 1.4 x 10(7) to 20 x 10(7) cell ml-1, the percentage of sporulating cells was found to decrease in parallel with the rate of acetate consumption. When the sporulation efficiency attained under the different experimental conditions was plotted as a function of the rate of acetate consumption, a linear correlation was found. Anabolic fluxes estimation revealed a decrease of the rate through gluconeogenic and glyoxylate pathways occurring during sporulation progression. The pattern of metabolic fluxes progressively evolved toward a predominance of more oxidative catabolic fluxes than those exhibited under growth conditions. The results obtained are discussed in terms of a characteristic pattern of metabolic fluxes and energetics, associated to the development of yeast sporulation.

Acetates↗
PubMed1995

Carbon and energetic uncoupling are associated with block of division at different stages of the cell cycle in several cdc mutants of Saccharomyces cerevisiae.

Cell proliferation arrest at 37 degrees C (restrictive temperature) of the cell division cycle (cdc) mutants of Saccharomyces cerevisiae cdc28, cdc35, cdc19, cdc21, and cdc17 was correlated with carbon and energy uncoupling. At 37 degrees C, cdc mutants diverted to biomass synthesis only 3 to 4% and 8 to 24% of the fluxes of carbon consumed and ATP obtained by catabolism, respectively, compared with 48 and 34% in the wild-type strain A364A. At the permissive temperature (25 degrees C), the wild type showed similar carbon and energy coupling indexes as at 37 degrees C. However, carbon and energy coupling indexes were two- to sevenfold higher at 25 degrees than at 37 degrees C in cdc mutants; e.g., at 25 degrees C two- to sevenfold higher amounts of carbon and ATP were directed to biomass production than at 37 degrees C. The wild-type strain exhibited a purely oxidative glucose catabolism at 37 degrees C (RQ approximately 1.0), while the cell proliferation arrest of cdc mutants at the same temperature was characterized by fermentative metabolism. At 37 degrees C, cdc mutants directed 50 to 60% of the carbon to ethanol production; 3 to 12% of the carbon was recovered as glycerol in cdc mutants as well as in the wild type. The proliferation arrest of the cell division cycle mutant cdc28 correlated with a significant decrease in the incorporation of radioactive precursors into DNA, RNA, and proteins. In the presence of 8-hydroxyquinoline, the wild-type strain underwent cell proliferation arrest and also exhibited metabolic uncoupling with bioenergetic and catabolic behavior similar to that of the cdc mutants at 37 degrees C. Experimental evidence obtained with cdc19, whose defective gene product is pyruvate kinase, suggests that the primary defect of cdc mutants correlates with a metabolically, highly uncoupled yeast cell. The results presented point to the existence of strong carbon and energy uncoupling together with cell division arrest exhibited by cdc mutants at the restrictive temperature. The degree of uncoupling appears to be tuned, at least in part, by the increase in flux of sugar catabolism through the ethanol fermentative pathway.

Carbon↗
PubMed1994

On the fractal nature of cytoplasm.

On the basis of a quantification of the fractal dimension, D, in micrographs of cytoskeleton components or microtrabecular lattice, we propose that the cellular cytoplasm can be described as a percolation cluster, a sort of 'random fractal'. Our hypothesis deals with: (i) the existence of the percolation threshold--a remarkable property of percolation processes; and (ii) the reactivity increase--when enzymes, or targets, and substrates, or effectors, coexist in the same topological dimension.

Catalysis↗
PubMed1994

Microtubular protein in its polymerized or nonpolymerized states differentially modulates in vitro and intracellular fluxes catalyzed by enzymes of carbon metabolism.

The fluxes through HK/G6PDH and PK/LDH coupled-enzymatic reactions were quantified in the presence of physiological concentrations (1-15 microM) of polymerized or non-polymerized microtubular protein (MTP) from rat brain and in a permeabilized yeast cell system. In vitro enzymatic fluxes were increased by either polymerized or nonpolymerized brain MTP mainly in the lower range of MTP concentration. At fixed MTP concentrations in the flux stimulatory range of HK/G6PDH (1 mg/ml MTP) or PK/LDH (0.4 mg/ml MTP), a hyperbolic and sigmoidal response to NADP and PEP, respectively, was detected. That dependence varied according to the polymeric status of MTP. The specificity of the phenomenon observed in vitro, was tested for the PK/LDH and HK/G6PDH enzymatic couples in the presence of neutral polymers such as glycogen (< or = 10 mg/ml), poly(ethylene glycol) (up to 10% w/w) or G-actin (< or = 1 mg/ml). In permeabilized Saccharomyces cerevisiae cells, the PK-catalyzed flux was sensitive to microtubule disruption by nocodazole (15 micrograms/ml). The HK/G6PDH system was not affected by nocodazole showing values of kinetic parameters close to those obtained in vitro in the presence of polymerized brain MTP. Indirect immunofluorescence with specific antibodies against tubulin allowed to confirm the microtubules disruption in the presence of nocodazole in permeabilized yeast cells under the same conditions in which enzymes were assayed intracellularly. The experimental evidence is in agreement with the observed phenomenon of increase in fluxes in the enzymatic reactions assayed to be specifically induced by MTP either in vitro or in situ. The results presented are discussed in terms of the assembly of large supramolecular structures as a supraregulatory mechanism of synchronization of systemic cellular processes such as metabolic fluxes.

Actins↗