The nucleocytoplasmic shuttling of the repressor Gal80p is known to play a pivotal role in the signal transduction process of GAL genetic switch of Saccharomyces cerevisiae (Peng, G., and Hopper, J. E. (2002) Proc. Natl. Acad. Sci. U. S. A. 99, 8548 -8553). We have developed a comprehensive model of this GAL switch to quantify the expression from the GAL promoter containing one or two Gal4p-binding sites and to understand the biological significance of the shuttling process. Our experiments show that the expression of proteins from the GAL promoter containing one and two binding sites for Gal4p is ultrasensitive (a steep response to a given input). Furthermore, the model revealed that the shuttling of Gal80p is the key step in imparting ultrasensitive response to the inducer. During induction, free Gal80p concentration is altered by sequestration, without any change in the distribution coefficient across the nuclear membrane. Furthermore, the estimated concentrations of Gal80p and Gal3p allow basal expression of ␣-galactosidase, but not ␤-galactosidase, from the GAL promoter containing one and two binding sites for Gal4p, respectively. Conversely, the expression from genes with two binding sites is more sensitive to inducer concentration as compared with one binding site. We show that autoregulation of Gal80p is coincidental to the autoregulation of Gal3p, and it does not impart ultrasensitivity. We conclude from our analysis that the ultrasensitivity of the GAL genetic switch is solely because of the shuttling phenomena of the repressor Gal80p across the nuclear membrane.

Silylamine reversible ionic liquids were designed to achieve specific physical properties in order to address effective CO₂ capture. The reversible ionic liquid systems reported herein represent a class of switchable solvents where a relatively non-polar silylamine (molecular liquid) is reversibly transformed to a reversible ionic liquid (RevIL) by reaction with CO₂ (chemisorption). The RevILs can further capture additional CO₂ through physical absorption (physisorption). The effects of changes in structure on (1) the CO₂ capture capacity (chemisorption and physisorption), (2) the viscosity of the solvent systems at partial and total conversion to the ionic liquid state, (3) the energy required for reversing the CO₂ capture process, and (4) the ability to recycle the solvents systems are reported.

Copper(I)-responsive fluorescent probes based on photoinduced electron transfer (PET) switching consistently display incomplete recovery of emission upon Cu(I) binding compared to the corresponding isolated fluorophores, raising the question of whether Cu(I) might engage in adverse quenching pathways. To address this question, we performed detailed photophysical studies on a series of Cu(I)-responsive fluorescent probes that are based on a 16-membered thiazacrown receptor ([16]aneNS3) tethered to 1,3,5-triarylpyrazoline-fluorophores. The fluorescence enhancement upon Cu(I) binding, which is mainly governed by changes in the photoinduced electron transfer (PET) driving force between the ligand and fluorophore, was systematically optimized by increasing the electron withdrawing character of the 1-aryl-ring, yielding a maximum 29-fold fluorescence enhancement upon saturation with Cu(I) in methanol and a greater than 500-fold enhancement upon protonation with trifluoroacetic acid. Time-resolved fluorescence decay data for the Cu(I)-saturated probe indicated the presence of three distinct emissive species in methanol. Contrary to the notion that Cu(I) might engage in reductive electron transfer quenching, femtosecond time-resolved pump-probe experiments provided no evidence for formation of a transient Cu(II) species upon photoexcitation. Variable temperature 1H NMR experiments revealed a dynamic equilibrium between the tetradentate NS3-coordinated Cu(I) complex and a ternary complex involving coordination of a solvent molecule, an observation that was further supported by quantum chemical calculations. The combined photophysical, electrochemical, and solution chemistry experiments demonstrate that electron transfer from Cu(I) does not compete with radiative deactivation of the excited fluorophore, and hence, that the Cu(I)-induced fluorescence switching is kinetically controlled.

a b s t r a c tSystems Biology has a mission that puts it at odds with traditional paradigms of physics and molecular biology, such as the simplicity requested by Occam's razor and minimum energy/maximal efficiency. By referring to biochemical experiments on control and regulation, and on flux balancing in yeast, we show that these paradigms are inapt. Systems Biology does not quite converge with biology either: Although it certainly requires accurate 'stamp collecting', it discovers quantitative laws. Systems Biology is a science of its own, discovering own fundamental principles, some of which we identify here. Crown

The photophysical properties of 1,3,5-triarylpyrazolines are strongly influenced by the nature and position of substituents attached to the aryl-rings, rendering this fluorophore platform well suited for the design of fluorescent probes utilizing a photoinduced electron transfer (PET) switching mechanism. To explore the tunability of two key parameters that govern the PET thermodynamics, the excited state energy ΔE 00 and acceptor potential E(A/A − ), a library of polyfluoro-substituted 1,3-diaryl-5-phenyl-pyrazolines was synthesized and characterized. The observed trends for the PET parameters were effectively captured through multiple Hammett linear free energy relationships (LFER) using a set of independent substituent constants for each of the two aryl rings. Given the lack of experimental Hammett constants for polyfluoro substituted aromatics, theoretically derived constants based on the electrostatic potential at the nucleus (EPN) of carbon atoms were employed as quantum chemical descriptors. The performance of the LFER was evaluated with a set of compounds that were not included in the training set, yielding a mean unsigned error of 0.05 eV for the prediction of the combined PET parameters. The outlined LFER approach should be well suited to design and optimize the performance of cation-responsive 1,3,5-triarylpyrazolines.

We have prepared and characterized a Cu(I)-responsive fluorescent probe, constructed using a large tetradentate, 16-membered thiazacrown ligand ([16]aneNS3) and 1,3,5-triaryl-substituted pyrazoline fluorophores. The fluorescence contrast ratio upon analyte binding, which is mainly governed by changes of the photoinduced electron transfer (PET) driving force between the ligand and fluorophore, was systematically optimized by increasing the electron withdrawing character of the 1-aryl-ring, yielding a maximum 50-fold fluorescence enhancement upon saturation with Cu(I) in methanol and a greater than 300-fold enhancement upon protonation with trifluoroacetic acid. The observed fluorescence increase was selective towards Cu(I) over a broad range of mono- and divalent transition metal cations. Previously established Hammett LFERs proved to be a valuable tool to predict two of the PET key parameters, the acceptor potential E(A/A−) and the excited state energy ΔE00, and thus to identify a set of pyrazolines that would best match the thermodynamic requirements imposed by the donor potential E(D+/D) of the thiazacrown receptor. The described approach should be applicable for rationally designing high-contrast pyrazoline-based PET probes selective towards other metal cations.

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AKR1B10 is an aldose reductase (AR) homologue overexpressed in liver cancer and various forms of that enzyme in carcinomas catalyze the reduction of anticancer drugs, potential cytostatic drug, and dl-glyceraldehyde but do not catalyze the reduction of glucose. Kinetic parameters for wild-type and C299S mutant AKR1B10 indicate that substitution of serine for cysteine at position 299 reduces the affinity of this protein for dl-glyceraldehyde and enhances its catalytic activity. Fibrates suppress peroxisome proliferation and the development of liver cancer in human. Here we report the potency of fibrate-mediated inhibition of the carbonyl reduction catalyzed by wild-type and C299S mutant AKR1B10 and compare it with known AR inhibitors. Wild-type AKR1B10-catalyzed carbonyl reduction follows pure non-competitive inhibition kinetics using zopolrestat, EBPC or sorbinil, whereas fenofibrate, Wy 14,643, ciprofibrate and fenofibric acid follow mixed non-competitive inhibition kinetics. In contrast, catalysis of reaction by the C299S AKR1B10 mutant is not inhibited by sorbinil and EBPC. Despite these differences, the C299S AKR1B10 mutant still manifests kinetics similar to the wild-type protein with other fibrates including zopolrestat, fenofibrate, Wy 14,346, gemfibrozil and ciprofibrate that show mixed non-competitive inhibition kinetics. The reaction of the mutant AKR1B10 is inhibited by fenofibric acid, but manifests pure non-competitive inhibition kinetics that are different from those demonstrated for the wild-type enzyme.