In vivo measurement of cytosolic and mitochondrial pH using a pH-sensitive GFP derivative in Saccharomyces cerevisiae reveals a relation between intracellular pH and growth The specific pH values of cellular compartments affect virtually all biochemical processes, including enzyme activity, protein folding and redox state. Accurate, sensitive and compartmentspecific measurements of intracellular pH (pH i ) dynamics in living cells are therefore crucial to the understanding of stress response and adaptation. We used the pH-sensitive GFP derivative 'ratiometric pHluorin' expressed in the cytosol and in the mitochondrial matrix of growing Saccharomyces cerevisiae to assess the variation in cytosolic pH (pH cyt ) and mitochondrial pH (pH mit ) in response to nutrient availability, respiratory chain activity, shifts in environmental pH and stress induced by addition of sorbic acid. The in vivo measurement allowed accurate determination of organelle-specific pH, determining a constant pH cyt of 7.2 and a constant pH mit of 7.5 in cells exponentially growing on glucose. We show that pH cyt and pH mit are differentially regulated by carbon source and respiratory chain inhibitors. Upon glucose starvation or sorbic acid stress, pH i decrease coincided with growth stasis. Additionally, pH i and growth coincided similarly in recovery after addition of glucose to glucose-starved cultures or after recovery from a sorbic acid pulse. We suggest a relation between pH i and cellular energy generation, and therefore a relation between pH i and growth. INTRODUCTIONMicrobes are able to adapt to a wide range of stressful environments such as deviant temperature, high or low osmotic pressure, oxidative stress and exposure to weak organic acids. The mechanisms by which they adapt to these environments are often poorly understood. To study these adaptive responses we rely on techniques that focus on various levels of cellular regulation, such as transcription profiles, protein levels and metabolic flux analysis. However, global physiological parameters such as intracellular pH (pH i ) affect nearly all processes in a living cell. pH i directly influences the redox state of the cell by influencing the NAD + /NADH equilibrium (Veine et al., 1998) and determines pH gradients necessary for transport over membranes (Goffeau & Slayman, 1981;Wohlrab & Flowers, 1982). Additionally, the effect of pH is very prominent in enzyme kinetics, as pH influences ionization states of acidic or basic amino acid side-chains and thereby influences the structure, solubility and activity of most, if not all, enzymes.The different organelles in the cell all maintain their own specific pH, which is used to define and maintain the processes associated with each organelle. Yeast vacuoles, for instance, are reported to have an acidic pH (Preston et al., 1989;Brett et al., 2005; Martínez-Muñoz & Kane, 2008). The proton gradient across the vacuolar membrane has been shown to be essential for the transport of various compounds (Nishimura et al., 1998;Ohsumi & Anraku, ...

The classic definitions of inulin and oligofructose are constructively criticized. It is observed that inulin cannot unequivocally be described as a polydisperse 1-kestose-based (GFn) beta (2-->1) linear fructan chain, but that inulin always contains small amounts of Fm and branched molecules. This review article describes the presence of inulin and oligofructose in common foodstuffs. Historical data on human consumption add an extra dimension. Modern analytical techniques (HPLC, LGC, HPAEC-PAD) are used to check the variety of data mentioned in the literature throughout the past century. Methods to determine inulin and oligofructose in natural foodstuffs (cereals, fruit, and vegetables) are optimized and used to determine the loss of inulin during storage and during preparation of the food. These findings allow quantification of the amount of inulin and oligofructose in the average daily western diet. The daily per capita intake is estimated to range from 1 to 10 g, depending on geographic, demographic, and other related parameters (age, sex, season, etc.). Inulin and oligofructose are not measured by classic methods of dietary fiber analysis and consequently are often not mentioned in food tables. Their significant contribution (1 to 10 g/d/per capita) to the dietary fiber fraction (recommended at 25 g/d/per capita) is not taken into account in any nutritional recommendations. In view of this, inulin and oligofructose deserve more attention, both in food composition tables and in diet or nutrition studies.

Recognition of lipids by proteins is important for their targeting and activation in many signaling pathways, but the mechanisms that regulate such interactions are largely unknown. Here, we found that binding of proteins to the ubiquitous signaling lipid phosphatidic acid (PA) depended on intracellular pH and the protonation state of its phosphate headgroup. In yeast, a rapid decrease in intracellular pH in response to glucose starvation regulated binding of PA to a transcription factor, Opi1, that coordinately repressed phospholipid metabolic genes. This enabled coupling of membrane biogenesis to nutrient availability.

Pacemaker and conduction system myocytes play crucial roles in initiating and regulating the contraction of the cardiac chambers. Genetic defects, acquired diseases, and aging cause dysfunction of the pacemaker and conduction tissues, emphasizing the clinical necessity to understand the molecular and cellular mechanisms of their development and homeostasis. Although all cardiac myocytes of the developing heart initially possess pacemaker properties, the majority differentiates into working myocardium. Only small populations of embryonic myocytes will form the sinus node and the atrioventricular node and bundle. Recent efforts have revealed that the development of these nodal regions is achieved by highly localized suppression of working muscle differentiation, and have identified transcriptional repressors that mediate this process. This review will summarize and reflect new experimental findings on the cellular origin and the molecular control of differentiation and morphogenesis of the pacemaker tissues of the heart. It will also shed light on the etiology of inborn and acquired errors of nodal tissues. (Circ Res. 2010;106:240-254.)Key Words: conduction system Ⅲ pacemaker Ⅲ sinus venosus Ⅲ atrioventricular canal Ⅲ development T he contractions of the heart are initiated and coordinated by electric signals from pacemaker tissues. At the entrance of the right atrium, sinus node (sinoatrial node [SAN]) myocytes generate the impulse to activate the atrial myocardium. After rapid propagation through the atria, the impulse is delayed in the atrioventricular node (AVN) and further propagated to the fast-conducting atrioventricular bundle (AVB), bundle branches (BB), and Purkinje fiber network, from which the mass of the ventricular working myocardium is activated. The components of the conduction system contain cardiomyocytes with pacemaker activity and other specific nodal properties that discriminate them from atrial and ventricular working myocardium (Figure 1). The SAN serves as the primary pacemaker, whereas the AVN and ventricular conduction system act as secondary (accessory) pacemakers to secure Original

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Realistic quantitative models require data from many laboratories. Therefore, standardization of experimental systems and assay conditions is crucial. Moreover, standards should be representative of the in vivo conditions. However, most often, enzyme-kinetic parameters are measured under assay conditions that yield the maximum activity of each enzyme. In practice, this means that the kinetic parameters of different enzymes are measured in different buffers, at different pH values, with different ionic strengths, etc. In a joint effort of the Dutch Vertical Genomics Consortium, the European Yeast Systems Biology Network and the Standards for Reporting Enzymology Data Commission, we have developed a single assay medium for determining enzyme-kinetic parameters in yeast. The medium is as close as possible to the in vivo situation for the yeast Saccharomyces cerevisiae, and at the same time is experimentally feasible. The in vivo conditions were estimated for S. cerevisiae strain CEN.PK113-7D grown in aerobic glucose-limited chemostat cultures at an extracellular pH of 5.0 and a specific growth rate of 0.1 h(-1). The cytosolic pH and concentrations of calcium, sodium, potassium, phosphorus, sulfur and magnesium were determined. On the basis of these data and literature data, we propose a defined in vivo-like medium containing 300 mM potassium, 50 mM phosphate, 245 mM glutamate, 20 mM sodium, 2 mM free magnesium and 0.5 mM calcium, at a pH of 6.8. The V(max) values of the glycolytic and fermentative enzymes of S. cerevisiae were measured in the new medium. For some enzymes, the results deviated conspicuously from those of assays done under enzyme-specific, optimal conditions.