Effect of macromolecular crowding on the kinetics of glycolytic enzymes and the behaviour of glycolysis in yeast

Water is involved in all aspects of biological activity, both as a solvent and as a reactant. It is hypothesized that intracellular water is in a highly structured state due to the high concentrations of macromolecules in the cell and that this may change the activity of intracellular enzymes due to altered binding affinities and allosteric regulations. Here we first investigate the kinetics of two glycolytic enzymes in artificially crowded aqueous solutions and show that crowding does indeed change their kinetics. Based on our kinetic measurements we propose a new model of oscillating glycolysis that instead of Michaelis-Menten or Monod-Wyman-Changeux kinetics uses the Yang-Ling adsorption isotherm introduced by G. Ling in the frame of the Association-Induction (AI) hypothesis. Using this model, we can reproduce previous experimental observations of the coupling of glycolytic oscillations and intracellular water dynamics, e.g., (i) during the metabolic oscillations, the latter variable oscillates in phase with ATP activity, and (ii) the emergence of glycolytic oscillations largely depends on the extent of intracellular water dipolar relaxation in cells in the resting state. Our results support the view that the extent of intracellular water dipolar relaxation is regulated by the ability of cytoplasmic proteins to polarize intracellular water with the assistance of ATP, as suggested in the AI hypothesis. This hypothesis may be relevant to the interpretation of many other biological oscillators, including cell signalling processes.Fil: Thoke, Henrik S.. University Of Southern Denmark; DinamarcaFil: Bagatolli, Luis Alberto. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Córdoba. Instituto de Investigación Médica Mercedes y Martín Ferreyra. Universidad Nacional de Córdoba. Instituto de Investigación Médica Mercedes y Martín Ferreyra; ArgentinaFil: Olsen, Lars F.. University Of Southern Denmark; Dinamarc

Impact of macromolecular crowding on the mesomorphic behavior of lipid self-assemblies

Using LAURDAN fluorescence we observed that water dynamics measured at the interface of DOPC bilayers can be differentially regulated by the presence of crowded suspensions of different proteins (HSA, IgG, Gelatin) and PEG, under conditions where the polymers are not in direct molecular contact with the lipid interface. Specifically, we found that the decrease in water dipolar relaxation at the membrane interface correlates with an increased fraction of randomly oriented (or random coil) configurations in the polymers, as Gelatin > PEG > IgG > HSA. By using the same experimental strategy, we also demonstrated that structural transitions from globular to extended conformations in proteins can induce transitions between lamellar and non-lamellar phases in mixtures of DOPC and monoolein. Independent experiments using Raman spectroscopy showed that aqueous suspensions of polymers exhibiting high proportions of randomly oriented conformations display increased fractions of tetracoordinated water, a configuration that is dominant in ice. This indicates a greater capacity of this type of structure for polarizing water and consequently reducing its chemical activity. This effect is in line with one of the tenets of the Association Induction Hypothesis, which predicts a long-range dynamic structuring of water molecules via their interactions with proteins (or other polymers) showing extended conformations. Overall, our results suggest a crucial role of water in promoting couplings between structural changes in macromolecules and supramolecular arrangements of lipids. This mechanism may be of relevance to cell structure/function when the crowded nature of the intracellular milieu is considered.Fil: Mangiarotti, Agustín. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Córdoba. Instituto de Investigación Médica Mercedes y Martín Ferreyra. Universidad Nacional de Córdoba. Instituto de Investigación Médica Mercedes y Martín Ferreyra; ArgentinaFil: Bagatolli, Luis Alberto. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Córdoba. Instituto de Investigación Médica Mercedes y Martín Ferreyra. Universidad Nacional de Córdoba. Instituto de Investigación Médica Mercedes y Martín Ferreyra; Argentina. Universidad Nacional de Córdoba. Facultad de Ciencias Químicas. Departamento de Química Biológica; Argentin

Lipids, membranes, colloids and cells: A long view

This paper revisits long-standing ideas about biological membranes in the context of an equally long-standing, but hitherto largely unappreciated, perspective of the cell based on concepts derived from the physics and chemistry of colloids. Specifically, we discuss important biophysical aspects of lipid supramolecular structure to understand how the intracellular milieu may constrain lipid self-assembly. To this end we will develop four lines of thought: first, we will look at the historical development of the current view of cellular structure and physiology, considering also the plurality of approaches that influenced its formative period. Second, we will review recent basic research on the structural and dynamical properties of lipid aggregates as well as the role of phase transitions in biophysical chemistry and cell biology. Third, we will present a general overview of contemporary studies into cellular compartmentalization in the context of a very rich and mostly forgotten general theory of cell physiology called the Association-Induction Hypothesis, which was developed around the time that the current view of cells congealed into its present form. Fourth, we will examine some recent developments in cellular studies, mostly from our laboratory, that raise interesting issues about the dynamical aspects of cell structure and compartmentalization. We will conclude by suggesting what we consider are relevant questions about the nature of cellular processes as emergent phenomena.Fil: Bagatolli, Luis Alberto. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Córdoba. Instituto de Investigación Médica Mercedes y Martín Ferreyra. Universidad Nacional de Córdoba. Instituto de Investigación Médica Mercedes y Martín Ferreyra; Argentina. Memphys - International And Interdisciplinary Research Network; Dinamarca. Universidad Nacional de Córdoba. Facultad de Ciencias Exactas, Físicas y Naturales. Departamento de Química. Cátedra de Química Biológica; ArgentinaFil: Stock, Roberto. Memphys - International And Interdisciplinary Research Network; Dinamarc

Scientific and technological policy and innovation dynamics in Brazil

Orientador: Renato Peixoto DagninoDissertação (mestrado) - Universidade Estadual de Campinas, Instituto de GeocienciasResumo: O final da década de 1990 marca uma mudança no direcionamento da PCT brasileira: o gasto público aumenta e é crescentemente direcionado às atividades inovativas empresariais. Essa mudança na PCT, quando analisada tendo por base a visão de pesquisadores que estudam a trajetória dessa política a partir da contribuição dos fundadores do Pensamento Latino-Americano em Ciência, Tecnologia e Sociedade, marca o que consideramos a sua quarta geração. Ela se caracterizaria por um aumento do esforço governamental para elevar a propensão das empresas locais a realizar P&D, que é identificada por eles e por esse pensamento como uma característica estrutural derivada da condição periférica de nossa sociedade. Adotando a perspectiva consagrada por aquele pensamento - a distinção entre política de C&T explícita e implícita - é possível encontrar exemplos da primeira modalidade que sustentam o argumento de que estaríamos em presença de uma quarta geração: o crescimento significativo da execução orçamentária do MCT e das suas agências, os Fundos Setoriais, a reativação do FUNTEC, os incentivos fiscais previstos na Lei de Inovação e na Lei do Bem e a subvenção econômica. Depois de descrever essa mudança, e mantendo aquela perspectiva, o trabalho penetra na esfera da política implícita de C&T para entender porque os indicadores disponibilizados pelas sucessivas edições da PINTEC (IBGE) parecem indicar que a política em curso não está logrando alterar significativamente a dinâmica tecnológica local. É baixo o grau de novidade dos produtos e processos introduzidos pelas empresas inovadoras, o dispêndio das empresas locais com atividades internas de P&D é muito menor do que o observado nos Países avançados, sendo que a parcela da receita líquida de vendas destinada à inovação por essas empresas - que já era relativamente pequena - diminuiu nos últimos anos. Outras evidências empíricas relativas ao cenário nacional e internacional sugerem a escassa probabilidade de uma alteração dessa situação. Entre elas, a importância que tem a realização de P&D na estratégia de inovação das empresas inovadoras: quatro vezes menor do que a aquisição de máquinas e equipamentos. A conclusão mais importante do trabalho corrobora o que aqueles autores fundadores e os pesquisadores contemporâneos têm afirmado. A baixa propensão a inovar (e, em particular, à realização de P&D) não é fruto de um "atraso cultural" dos empresários ou da falta de instrumentos que induzam o empreendedorismo e a competitividade. Ele é uma resposta racional das empresas locais ao que foi denominado política implícita de C&T, que reproduz a nossa condição periférica e mantém os sinais de mercado que não as estimulam. E é pouco provável que a quarta geração da PCT, a menos que seu aprofundamento a transforme em algo ainda mais incoerente com as outras demandas - que não as empresariais - que deveria atender esta política, possa neutralizar os obstáculos estruturais existentes em nossa sociedade; os quais apesar de há muito conhecidos, são atualmente bem pouco lembrados pelo pensamento oficial.Abstract: The late 1990's are characterized by a change on the Brazilian Science and Technology Policy (STP): the public expenditure increases and it is been increasingly focused towards innovative entrepreneurs activities. This shift on the STP when analyzed through the conceptions of the scholars who study this trajectory under the contributions made by the founders of the Latin-American Thought on Science Technology and Society school marks its fourth generation. This policy is characterized by an increase of the governmental efforts to elevate local companies' propensity to realize R&D which is understood by them and by this school of thought as a structural characteristic of our society peripheral condition. Taking this perspective - the distinction between the explicit and implicit S&T Policy - it is possible to find examples of the first model of policy which sustains the argument that we would be in the presence of a fourth generation: the substantial increase of MCT (Brazilian Science and Technology Ministry) and its agencies budget implementation, the existence of sectored funds, the reactivation to the FUNTEC, the tax incentives contained in the Innovation Law and in the Law of Good and the economic subsidy. After describing this change, and keeping that perspective, the work enters into the sphere of the implicit S&T policy to understand why the indicators provided by successive editions of PINTEC (IBGE) seems to indicate that the current policy is not managing to alter the local technology dynamics significantly. Its low the level of novelty on products and processes introduced by innovative companies and the spending of local firms with internal activities of R&D are much smaller than that seen in advanced countries, even worst is that the amount of net revenues from sales to innovation by these companies - which was already relatively small - declined in the recent years. Other empirical evidences, on the national and international scenario, show low likelihood of a change in that situation. Among them is the importance of conducting R&D in the strategy of innovation in innovative companies which in this case is four times less than the purchase of machinery and equipment. The most important conclusion of the work confirms what those founder authors and contemporary researchers have said. The low propensity to innovate (and, in particular, to carry out R&D) is not the result of the entrepreneurs' "cultural backwardness" or the lack of instruments which lead to entrepreneurship and competitiveness. It is a rational response of local companies to what was called implicit C&T policy that reproduces our peripheral condition and maintains the signals of the market which do not stimulate them. And it is unlikely that the fourth generation of the PCT, unless its deepening turn into something even more inconsistent with the other demands - not the business - which should take this policy, could neutralize the structural barriers that exist in our society, and which are currently very little remembered by the official thinking.MestradoMestre em Política Científica e Tecnológic

Coupled response of membrane hydration with oscillating metabolism in live cells: An alternative way to modulate structural aspects of biological membranes?

We propose that active metabolic processes may regulate structural changes in biological membranes via the physical state of cell water. This proposition is based on recent results obtained from our group in yeast cells displaying glycolytic oscillations, where we demonstrated that there is a tight coupling between the oscillatory behavior of glycolytic metabolites (ATP, NADH) and the extent of the dipolar relaxation of intracellular water, which oscillates synchronously. The mechanism we suggest involves the active participation of a polarized intracellular water network whose degree of polarization is dynamically modulated by temporal ATP fluctuations caused by metabolism with intervention of a functional cytoskeleton, as conceived in the long overlooked association-induction hypothesis (AIH) of Gilbert Ling. Our results show that the polarized state of intracellular water can be propagated from the cytosol to regions containing membranes. Since changes in the extent of the polarization of water impinge on its chemical activity, we hypothesize that metabolism dynamically controls the local structure of cellular membranes via lyotropic effects. This hypothesis offers an alternative way to interpret membrane related phenomena (e.g., changes in local curvature pertinent to endo/exocytosis or dynamical changes in membranous organelle structure, among others) by integrating relevant but mostly overlooked physicochemical characteristics of the cellular milieu.Fil: Bagatolli, Luis Alberto. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Córdoba. Instituto de Investigación Médica Mercedes y Martín Ferreyra. Universidad Nacional de Córdoba. Instituto de Investigación Médica Mercedes y Martín Ferreyra; ArgentinaFil: Stock, Roberto P.. International and Interdisciplinary Research Network; DinamarcaFil: Olsen, Lars F.. University Of Southern Denmark; Dinamarc

A General Theory of Non-equilibrium Dynamics of Lipid-protein Fluid Membranes

We present a general and systematic theory of non-equilibrium dynamics of multi-component fluid membranes, in general, and membranes containing transmembrane proteins, in particular. Developed based on a minimal number of principles of statistical physics and designed to be a meso/macroscopic-scale effective description, the theory is formulated in terms of a set of equations of hydrodynamics and linear constitutive relations. As a particular emphasis of the theory, the equations and the constitutive relations address both the thermodynamic and the hydrodynamic consequences of the unconventional material characteristics of lipid-protein membranes and contain proposals as well as predictions which have not yet been made in already existed work on membrane hydrodynamics and which may have experimental relevance. The framework structure of the theory makes possible its applications to a range of non-equilibrium phenomena in a range of membrane systems, as discussions in the paper of a few limit cases demonstrate.Comment: 22 pages, 2 figures, minor changes and addition

Validación de un método alternativo para la conservación de bacterias

La Cátedra de Microbiología de la Facultad de Ciencias Agrarias, UNCuyo, posee una Colección de Cultivos Microbianos, que alberga microorganismos utilizados en docencia, servicios e investigación. Existe un método internacionalmente reconocido, CRYOBANK, en el que los microorganismos son conservados por congelación, en viales con “perlitas" sobre las cuales éstos son adheridos. Presenta la ventaja de que ante la necesidad de activar un microorganismo una perla se siembra, en medio líquido o sólido, sin descongelar todo el vial; pero tiene la desventaja de su costo y su venta en cantidad no menor a 100 viales. Si bien en la práctica es frecuente la congelación utilizando mostacillas, en lugar de los viales comerciales, no hay datos sobre sus resultados. El objetivo de la tesis fue evaluar la efectividad de un método de conservación microbiano alternativo respecto al CRYOBANK. Para llevarlo a cabo se seleccionaron 12 cepas bacterianas, se activaron, se controló pureza y viabilidad, se conservaron por ambos métodos y al cabo de 24 horas, 1, 3, 6 y 15 meses se evaluó vialidad, pureza, porcentaje de recuperación y estabilidad de cada una. A los 15 meses el 83 % de las cepas conservadas no presentaron diferencias significativas en el recuento medio para ambos métodos. Todas se mantuvieron estables, puras y presentaron un porcentaje de recuperación mayor al 50 % a los 6 meses y el 67 % de ellas a los 15 meses. Por lo que el método de conservación alternativo permite preservar bacterias, al menos por el periodo de tiempo evaluado.Fil: Bagatolli Garófoli, Carolina Daniela. Universidad Nacional de Cuyo. Facultad de Ciencias Agrarias

The cell as a gel: materials for a conceptual discussion

Recent results from our laboratory support the view that the intracellular milieu cannot be treated as a homogeneous dilute system and, more importantly, reveal for the first time a dynamical coupling between intracellular water and an active metabolic process involving fluctuations in ATP concentration. These results are difficult to understand in light of the premises that currently underpin the description of the function of cellular systems, e.g. van’t Hoff’s ideal solution theory, diffusion and mass action kinetics. Particularly, they emphasize the need to incorporate features of the cell interior that have been largely overlooked in the dominant model of the cell, such as crowding and limited availability of free water. This article discusses this problem by reconsidering an alternate view, called the association-induction hypothesis, which emphasizes the relevance of emergent properties of the cell cytosol during cellular function. This hypothesis provides a very reasonable theoretical framework to explain recently reported observations about the dynamical coupling of mechanochemical (i.e. viscoelastic) properties of the cell cytoplasm and cellular chemical transformations (metabolism).Los resultados experimentales obtenidos recientemente en nuestro laboratorio apoyan la idea que el medio intracelular no puede ser tratado como un sistema homogéneo (o solución diluida), revelando además por primera vez un acoplamiento dinámico entre el comportamiento colectivo del agua intracelular y un proceso metabólico activo que muestra fluctuaciones en la concentración de ATP. Estos nuevos resultados -que son difíciles de interpretar en base a los supuestos más generalmente utilizados para interpretar las bases fisicoquímicas de la fisiología de los sistemas celulares (p.ej. teoría de las soluciones ideales de van't Hoff, difusión, y cinética de acción de masas)- subrayan la necesidad urgente de incorporar características importantes del interior celular, tales como el hacinamiento molecular y la escasa disponibilidad de agua libre. Este artículo analiza críticamente este problema considerando una hipótesis alternativa, llamada hipótesis de asociación-inducción, la cual hace hincapié en la importancia de las propiedades emergentes del citosol durante la función celular. Esta hipótesis proporciona un marco teórico razonable para explicar nuestras observaciones, particularmente el acoplamiento dinámico entre las propiedades mecanoquímicas (o viscoelásticas) del citoplasma celular y las transformaciones químicas (metabolismo) en el interior celular.Sociedad Argentina de Fisiologí

Análisis filosófico sobre la concepción neurocientífica de los rasgos autistas: aportes desde el normativismo vital de Georges Canguilhem

Taking Georges Canguilhem’s 1943 book The normal and the pathological as a starting point, this article explores the ways in which the neurosciences define, validate, and legitimize the existence of autistic traits as a subclinical expression of autism. The general hypothesis is that different assumptions based on a naturalistic perspective of health and disease have become consolidated in the specialized literature. Such assumptions include that behaviors should be explained strictly in biological terms, that there is an objective and statistical parameter of normality, and that individuals’ behaviors can be analyzed independently of their context. Based on some aspects of Canguilhem’s arguments regarding health, normality, and normativity, we analyze the ways in which the neurosciences assign a quasi-pathological quality to the descriptive notion of autistic traits. It is possible to conclude that this process involves certain assumptions taken in an aprioristic and uncritical manner, which inhibits the discussion of key aspects associated with the nature of autism and that end up pathologizing differences between people.A partir de la obra Lo normal y lo patológico de Georges Canguilhem, publicada en 1943, se explora cómo las neurociencias definen y legitiman, en la actualidad, la existencia de los rasgos autistas como expresión subclínica de autismo. La hipótesis general del presente trabajo es que en la bibliografía especializada de este ámbito parecen consolidarse diferentes supuestos asumidos en el marco de una perspectiva naturalista de la salud. Tales supuestos refieren a que los comportamientos deben ser explicados estrictamente en términos biológicos, que existe un parámetro de normalidad de carácter objetivo y estadístico, y que las conductas de los individuos pueden ser analizadas separadamente del contexto. Retomando algunos aspectos de la propuesta de Canguilhem sobre salud, normalidad y normatividad, se analiza de qué manera las neurociencias otorgan una cualidad cuasi-patológica a una noción descriptiva como la de rasgos autistas. A nuestro entender, este proceso involucra la asunción apriorística y acrítica de supuestos que impiden poner en discusión aspectos elementales asociados a la naturaleza del autismo y que conducen a patologizar la diferencia entre personas