Large shallow lake response to anthropogenic stressors and climate change: Missing macroinvertebrate recovery after oligotrophication (Lake Balaton, East-Central Europe).

East-Central Europe's largest shallow lake, Balaton, experienced strong eutrophication in the 1970-80s, followed by water quality improvement and oligotrophication by 2010 CE. Recently however, repeated cyanobacterial blooms occurred and warned that internal P-recycling can act similarly to external P load, therefore we need a better understanding of past water level (WL) and trophic changes in the lake. In this study we discuss the last 500-yr trophic, WL and habitat changes of the lake using paleoecological (chironomids, pollen) and geochemical (sediment chlorophyll, TOC, TS, TN, C/H ratio, major and trace element) methods. We demonstrate that the most intensive and irreversible change in the macroinvertebrate fauna occurred during the period of economic boom between the First and Second World War (∼1925-1940 CE), when large-scale built-in and leisure use of the lake has intensified. At that time, the Procladius-Microchironomus-Stempellina dominated community transformed to Procladius-Chironomus plumosus-type-Microchironomus community that coincided with land use changes, intensified erosion and water-level regulation in the lake with the maintenance of year-round high WL. This was followed by the impoverishment and population size decrease of the chironomid fauna and Procladius dominance since 1940 CE, without any recovery after 1994 CE despite the ongoing oligotrophication. Accelerated rate of change and turnover of the fauna was connected to an increase in the benthivorous fish biomass and eutrophication. The basin lost almost completely its once characteristic Stempellina species between 1927 and 1940 CE due to trophic level increase and seasonal anoxia in the Szemes Basin. Reference conditions for ecosystem improvement were assigned to 1740-1900 CE. We conclude that in spite of the ongoing oligotrophication, the re-establishment of the Procladius-Microchironomus-Stempellina assemblage is hampered, and requires fish population regulation.

Optical Gaps in Pristine and Heavily Doped Silicon Nanocrystals: DFT versus Quantum Monte Carlo Benchmarks.

We present a time-dependent density functional theory (TDDFT) study of the optical gaps of light-emitting nanomaterials, namely, pristine and heavily B- and P-codoped silicon crystalline nanoparticles. Twenty DFT exchange-correlation functionals sampled from the best currently available inventory such as hybrids and range-separated hybrids are benchmarked against ultra-accurate quantum Monte Carlo results on small model Si nanocrystals. Overall, the range-separated hybrids are found to perform best. The quality of the DFT gaps is correlated with the deviation from Koopmans' theorem as a possible quality guide. In addition to providing a generic test of the ability of TDDFT to describe optical properties of silicon crystalline nanoparticles, the results also open up a route to benchmark-quality DFT studies of nanoparticle sizes approaching those studied experimentally.

The dynamic basis of energy transduction in enzymes.

The most important idea underlying our treatment herein is the unity of the enzyme molecule and the medium. Appreciation of this relationship is vital, if enzymology is to graduate from its present reductionistic status to a more holistic posture. Enzymes are biological entities firstly, and isolated objects of physicochemical analysis secondly. Perhaps the most crucial 'biological lesson', particularly apropos of enzymes in intermediary metabolism, concerns the 'cytosociology' of enzyme action in vivo [94,128]. The natural habitat of many enzymes in the living cell is far different from that in bulk aqueous solution in vitro. In order to obtain a real grasp of the nature of enzyme function, one must ultimately couch enzymology in concepts emerging from contemporary cell biology [95]. Notwithstanding, analysis precedes synthesis; and one must needs begin with the individual enzyme molecule. The trenchant efforts of the physical chemist and the organic chemist have produced a wealth of information on the nature of the binding and catalytic events at the enzyme active site. While it is not yet possible to explain precisely the complete sequence of events in the catalytic process, nevertheless, the basic mechanisms by which enzymes effect catalysis (i.e., reduce activation energy) now seem apparent [81,129]. The new frontier is to be found, in exploring the dynamic role of the protein matrix [17]. Not only does the protein provide the 3-D scaffolding for active-site processes, but, more importantly, it serves as the local solvent for the bound chemical subsystem. Thus, the dynamical aspects of enzyme catalysis (for thermally based systems) must arise from the fluctuational properties of the protein molecule. This notion is the common denominator in all of the models in subsection IIC. It is the anisotropic nature of this fluctuational behavior, which would characterize the energy-transduction phenomenon leading to localized catalytic events at the active-site. In Section III we attempted to show that all of the various enzyme models contribute pieces to a single, all-embracing jig-saw puzzle. Some models focus on the dynamical properties of the protein per se, whereas others deal with the stochastic aspects of protein-solvent interaction. The two approaches are complementary, as are mutually interlocking pieces of a puzzle. The ultimate picture depicted by this 'jig-saw puzzle' is still somewhat vague--owing to the present paucity of empirical information on protein motions.(ABSTRACT TRUNCATED AT 400 WORDS)

Viscosity dependence of acrylamide quenching of ribonuclease T1 fluorescence. The gating mechanism.

The structural regulation of the access of acrylamide molecules, as quenchers, to the buried tryptophans of a protein can be modelled by a simple gate concept. Such a gate, when open, allows transient exposure of the fluorophore to the quencher molecule in solution. We have previously shown that the observed viscosity dependence of acrylamide quenching process in ribonuclease T1 (RNAse T1) is not reconcilable with the gating mechanism. However, on that occasion, we neglected the effect of changes in the activity of the quencher molecule and the possible presence of static quenching. The experimental observation of a considerable contribution by static quenching and the realization that static quenching might produce dramatic effects in steady state measurements led us to reexamine the question. It is shown that in a gating model the static component can also influence the apparent dynamic quenching. In this paper, we present derived equations for the gated quenching mechanism including possible contributions from the static component. We also carefully remeasured the acrylamide quenching of RNAase T1 as a function of increasing glycerol concentration. Computer simulations were carried out to compare the experimental data set to the generalized model. We reach the conclusion that even the new, quite complex equations fail to predict the qualitative and quantitative features of the observed quenching experiments. We arrived at the conclusion that the fluorophore is never the target of the quencher molecules in solution.

The effect of transmembrane potential on the dynamic behavior of cell membranes.

The relationship between transmembrane potential and lipid dynamics in the cytoplasmic membrane of mouse thymus cells has been investigated. Changes of transmembrane potential was followed by measuring the fluorescence emission of the anionic dye, bis-(1,3-dibutylbarbiturate)trimethine oxonol (diBa-C4-(3)). Assessment of lipid fluidity was carried out applying three fluorescent lipid probes, 1-[4-(trimethylammonium)phenyl]-6-phenyl-1,3,5-hexatriene (TMA-DPH), 12-(9-anthroyloxy)stearic acid (12-AS) and 1,6-diphenyl-1,3,5-hexatriene (DPH) used to monitor different structural regions of the bilayer. The fluorescence anisotropy of these probes was measured as a function of temperature at two values of transmembrane potential. In the case of DPH it proved to depend on the membrane potential in the higher temperature range (above 28 degrees C), while no such a dependence could be observed for DPH below this temperature range and for TMA-DPH and 12-AS in between 20 and 37 degrees C. These data suggest that changes in transmembrane potential are accompanied with some local alteration in membrane lipid dynamics and/or structure.

Segmental mobility in glycogen phosphorylase b.

The dynamics and structuredness of the pyridoxal 5'-phosphate-binding region in glycogen phosphorylase b (EC 2.4.1.1) has been investigated with different techniques of fluorescence spectroscopy. Fluorescence polarization data of the thermal Perrin plot indicate some mobility in the cofactor binding site, while the isothermic measurements (at 20 degrees C, in high-viscosity solvents) demonstrate that the mobile unit carrying the emission oscillator is practically insensitive to the external viscosity. Characteristics of the thermal Perrin plots obtained for both native and reduced phosphorylase b can be interpreted either as a freely moving cofactor in a medium of high viscosity (0.3 P) or as the motion of a unit larger than a lysine-bonded pyridoxal 5'-phosphate in a medium with the viscosity of water. Data for acrylamide quenching and time-resolved fluorescence measurements suggest that the latter interpretation should valid. These data also suggest a tightly packed microenvironment around the pyridoxal moiety.

Coupling between external viscosity and the intramolecular dynamics of ribonuclease T1: a two-phase model for the quenching of protein fluorescence.

Our recent equilibrium dialysis studies showed that proteins are able to interact preferentially with acrylamide (Punyiczki et al. (1993) Biophys. Chem. 47, 9-19). The presence of considerable amounts of acrylamide--albeit weakly bound--in the protein volume, coupled with the failure of a simple gating model of quenching to rationalise viscosity dependence of the quenching of tryptophan (Trp) fluorescence in Ribonuclease T1 (RNase T1) has prompted us to explore a new model, the two-phase model for quenching. According to this model, the dynamic quenching is accomplished by quencher molecules already in the protein phase at the moment of excitation. Some of the molecules may, at this moment, form an encounter complex with the fluorophore and thus be responsible for the observed static contribution. We use the rate equation derived from our model to study the viscosity dependence of acrylamide quenching of Trp fluorescence in RNase T1. The model allows us to separate co-solvent effects: the chemical effect on the protein and on the distribution of quencher molecules between the bulk and the protein phases and, further, the viscosity effect due to coupling between the bulk viscosity and the local friction affecting intramolecular fluctuations of the protein matrix. We express local friction in terms of bulk viscosity, eta, and a coupling constant kappa (friction = eta kappa). Addition of glycerol up to 65% is characterised by a kappa of 0.50. The viscosity dependence of the apparent bimolecular quenching constant is a combination of two compensating effects: changes in chemical activity and changes in patterns of structural fluctuations.

Characterization of living normal and leukemic mouse lymphocytes by fluorescein diacetate.

The esterases activity of normal and acute leukemic mouse lymphocytes and that of their homogenates was investigated using fluorescein diacetate (FDA) as a fluorogenic substrate. The activity proved to be the same for the two cell populations as well as for the homogenates prepared from them. In cell suspensions, having different osmolalities, the rate of FDA hydrolysis decreased significantly with the increasing osmolality only in the case of intact leukemic lymphocytes. changes in the membrane and cytoplasmic viscosity caused by increased or decreased environmental osmolality of cell suspensions occurred in the same direction and to the same extent for both normal and leukemic cells. Fluorescein, the fluorescent product of the hydrolysis, accumulates in leukemic lymphocytes, whereas it easily effluxes form normal lymphocytes. A flow microfluorimetry analysis of the cell population revealed that the fluorescein content of large leukemic lymphocytes was three times higher than that of small, normal ones. The observed differences specific for leukemic lymphocytes might be useful in detecting leukemic transformation in an early stage of acute lymphoid leukemia.

Phase-fluorimetry study on dielectric relaxation of human serum albumin.

The dielectric relaxation (DR) of human serum albumin (HSA) was studied by the method of phase-fluorometry. The protein environment of the single tryptophan in HSA shows a relatively low-speed DR of sub-ns characteristic time. This relaxation can be measured as a decaying red-shift of the time-resolved fluorescence emission spectra. The details of calculations of time-emission matrices (TEM) and comparison to the fluorescence data of the reference solution of N-acetyl-L-tryptophanamide (NATA) are also presented.

Phase-fluorometry study on dielectric relaxation of acrylodan-labeled human serum albumin.

Dielectric relaxation (DR) of acrylodan-labeled human serum albumin (HSA/AC) was studied by phase-fluorometry. A non-monoexponential behavior of both the total fluorescence--and the DR decays has been found. The protein environment of the fluorescent marker shows DR times ranging from the pico to nanosecond timescale. In fluorescence emission decays measured on the red side of the fluorescence spectrum a time constant (<10 ps) affected by a negative preexponential was found supporting the existence of DR of the excited states.

Viscosity and transient solvent accessibility of Trp-63 in the native conformation of lysozyme.

We have measured the rates of isotope exchange at the nitrogen of the indole ring of Trp-63 of lysozyme and of L-tryptophan as a function of solution viscosity. We have used two cosolvents, glycerol and ethylene glycol, to modify the relative viscosity. We have derived the appropriate kinetic equations for the alternative possibilities that the exchange takes place either in solution or in the intact protein matrix. Because we chose to study the proton-catalyzed exchange reaction, the rate of it is not expected to be diffusion-limited. We confirmed this by measuring the exchange from tryptophan. These results and the known effects of glycerol and ethylene glycol on the solvation of indole allow us to predict that if the exchange reaction takes place in a protein matrix the effects of the two cosolvents when compared under isoviscous conditions should be identical. This is what we find for Trp-63 in lysozyme at 15, 20 and 26 degrees C. The slope of the linear plot of log k vs. log relative viscosity is 0.6. This strongly supports a model for conformational fluctuations where transient solvation takes place without major changes in protein folding. The most interesting feature of our findings is the fact that a slow reaction admittedly not diffusion-limited shows, when taking place in a protein matrix, a linear dependence on solution viscosity. We suggest that what we observe is the effect of damping of movement of the side chain expressed as a change in the friction along the reaction coordinate in the corresponding phase space. The presence of such effects stresses the validity and usefulness of Kramers model of rate processes for reactions taking place in a protein matrix. Such behavior is predicted by several of the recently proposed general mechanisms of enzyme catalysis.

Protein dynamics and fluorescence quenching.

There are both theoretical and experimental data which strongly suggest that the intramolecular dynamics of the protein matrix play an important role in protein functions. The interrelationship between theory and experiments is rather weak mainly because of the lack of relevant experimental methods and (model-dependent) misinterpretation of experimental data. We give a short account of a few fluorescence-quenching techniques that can provide adequate information concerning protein dynamics provided that the experimental data sets are correctly processed.

Protein flexibility as revealed by fluorescence resonance energy transfer: an extension of the method for systems with multiple labels.

The temperature profile of the normalized fluorescence resonance energy transfer efficiency is capable of monitoring the relative change of flexibility and/or conformational state of macromolecules [Biochemistry 23 (1984) 3403]. The method described earlier for one donor-one acceptor systems is extended to multiple fluorophore systems when the energy transfer occurs between either one donor-m acceptors, or n donors-one acceptor or n donors-m acceptors (where n and m are integer values). It is shown that the normalized energy transfer efficiency obtained for systems containing multiple labels is a linear combination of the normalized transfer efficiency assigned to individual donor-acceptor pairs of the system, thus its temperature profile is capable of monitoring the change of intramolecular flexibility and/or conformational state.

Characterization of intracellular calcium oscillations induced by extracellular nucleotides in HEp-2 cells.

The effect of extracellular nucleotides on the cytosolic calcium concentration of fluo-3-loaded HEp-2 cells was examined using confocal microscopy. Extracellular ATP and UTP at micromolar concentration induced cytosolic calcium oscillations in 42-66% of the cells. Oscillations were usually sinusoid and their frequency depended only slightly on agonist concentration. Oscillations developed in calcium-free medium but were diminished by depletion of intracellular calcium stores with thapsigargin, indicating periodic calcium release from internal stores. Inhibition of phospholipase C with U73122 prevented the development of oscillations, while ryanodine did not abolish the response to extracellular nucleotides. Activation of protein kinase C with 4beta-phorbol 12-myristate 13-acetate also prevented the development of oscillations. These results indicate that extracellular nucleotides induce periodic calcium release from inositol 1,4,5-trisphosphate-sensitive pools in HEp-2 cells and that the inhibitory effect of protein kinase C on the phosphatidylinositol signaling pathway can contribute to the development of intracellular calcium oscillations.

Fluorescence quenching of the tryptophan emission from the F- and G-forms of actin.

The parameters characterizing the quenching of fluorescence emitted by the four tryptophans of actin were measured by time resolved techniques in the monomeric (G) and the polymerized (F) forms of the protein. Acrylamide as a neutral and cesium ions as positively charged quenchers were used to characterize the subdomain 1, which contains all the four tryptophan residues. Use of acrylamide did not reveal any difference between the G- and F-forms, cesium, however, did so. The value of the quenching rate constant, k+, is significantly higher for the F-form than that for the G-form. This difference is present independently of the model (discrete or continuous lifetime distribution) used to process the data. These results are compatible with the conclusion that the charge distribution of the microenvironment around at least one of the four tryptophans is changed. This means that this region becomes more attracted to the cesium ion as a result of transition from the G- to the F-form.

Alteration of the intramolecular dynamics of glycogen phosphorylase b by allosteric ligands.

Phosphorylase b (E.C. 2.4.1.1), prepared from rabbit skeletal muscle, was used to study whether the binding of allosteric ligands modifies the intramolecular dynamics of the protein matrix. Protein dynamics were monitored through the fluorescence and phosphorescence parameters of the 12 tryptophan (Trp) residues (one monomer) of the enzyme. The phosphorescence lifetime was measured at room temperature both in the absence and the presence of ligands. The addition of an allosteric inhibitor (ATP) decreased the lifetime, while the presence of activator (AMP) and/or substrate (G-1-P) had no detectable effect. The lifetime data allow us to conclude that the environment of the buried tryptophans becomes more flexible upon the binding of ATP, while the other ligands did not induce such change. The ATP-induced perturbation was also examined by the quenching of Trp fluorescence by acrylamide. The quenching parameters did not show any change, suggesting that the effect of ATP is localized to the vicinity of the phosphorescent Trp residues.

Application of a molecular enzyme kinetic model for aging cells and tissues.

According to the membrane hypothesis of aging (MHA), cellular senescence is attributable to a life-long, cross-linking action of oxygen-free radicals in the cell plasma membrane, resulting in a continuous decrease of the passive ion permeabilities. The consequent increase in the intracellular potassium content is accompanied by a considerable condensation of the intracellular mass (i.e., by loss of water). MHA suggested that an age-dependent increase in the physical density of the intracellular mass can underly the well-known age-dependent decreases of the macromolecular synthetic processes, the enzymic turnover rates, etc. MHA was partly based on a molecular enzyme kinetic model (MEKM) suggesting that environmental factors can substantially influence the enzyme catalysis and regulation through collisional coupling. However, the possible quantitative ranges of alterations in enzyme activities have not been estimated. This paper concludes, using principal features of the two models, that known age-dependent changes in the membrane lipid fluidity and intracellular density may result in even a 10-fold overall decrease in the enzyme activities (characterized by kcat and k-1) during the life.

The use of a new fluorescent viscosity indicator to study the relationship between transmembrane potential and membrane dynamics of lymphocytes.

A new fluorescent dye, polymethine derivative 4501 U, was applied to label the cytoplasmic membrane of mouse lymphocytes to investigate whether the membrane lipid dynamics is related to the transmembrane potential. The dye was found not to alter the cell viability. The dye is localized close to the external surface of the membrane, and its spectroscopic characteristics make it possible to obtain information on membrane fluidity using the intensity ratio of its two emission peaks. In contrast to recent data indicating that transmembrane potential alters the membrane lipid dynamics (as revealed by anisotropy studies with diphenyl hexatriene) 4501 U does not show such a relationship. The investigations presented support the hypothesis that the change in the transmembrane potential affects the dynamics/structure of the membrane region only at the interface between the two lipid layers.