THE KINETICS OF DARK ADAPTATION

1. Data are presented for the dark adaptation of four species of animals. They show that during dark adaptation the reaction time of an animal to light of constant intensity decreases at first rapidly, then slowly, until it reaches a constant minimum. 2. On the assumption that at all stages of adaptation a given response to light involves a constant photochemical effect, it is possible to describe the progress of dark adaptation by the equation of a bimolecular reaction. This supposes, therefore, that dark adaptation represents the accumulation within the sense cells of a photosensitive material formed by the chemical combination of two other substances. 3. The chemical nature of the process is further borne out by the fact that the speed of dark adaptation is affected by the temperature. The velocity constant of the bimolecular process describing dark adaptation bears in Mya a relation to the temperature such that the Arrhenius equation expresses it with considerable exactness when µ = 17,400. 4. A chemical mechanism is suggested which can account not only for the data of dark adaptation here presented, but for many other properties of the photosensory process which have already been investigated in these animals. This assumes the existence of a coupled photochemical reaction of which the secondary, "dark" reaction is catalyzed by the products of the primary photochemical reaction proper. This primary photochemical reaction itself is reversible in that its main products combine to form again the photosensitive material, whose concentration controls the behavior of the system during dark adaptation.     

Confining domains lead to reaction bursts: reaction kinetics in the plasma membrane

Confinement of molecules in specific small volumes and areas within a cell is likely to be a general strategy that is developed during evolution for regulating the interactions and functions of biomolecules. The cellular plasma membrane, which is the outermost membrane that surrounds the entire cell, was considered to be a continuous two-dimensional liquid, but it is becoming clear that it consists of numerous nano-meso-scale domains with various lifetimes, such as raft domains and cytoskeleton-induced compartments, and membrane molecules are dynamically trapped in these domains. In this article, we give a theoretical account on the effects of molecular confinement on reversible bimolecular reactions in a partitioned surface such as the plasma membrane. By performing simulations based on a lattice-based model of diffusion and reaction, we found that in the presence of membrane partitioning, bimolecular reactions that occur in each compartment proceed in bursts during which the reaction rate is sharply and briefly increased even though the asymptotic reaction rate remains the same. We characterized the time between reaction bursts and the burst amplitude as a function of the model parameters, and discussed the biological significance of the reaction bursts in the presence of strong inhibitor activity.     

Topological information content and chemical reactions

The difference of the topological information content of two reacting molecules and that of their reaction products is calculated for several topological types of chemical reactions, illustrating the influence of the structure of the reagents and of the reaction product. It is shown that the change in the topological information content in a chemical reaction can be positive as well as negative, depending on the way the reagents approach each other and thus on the reaction product formed. A quantitative measure of structural specificity is introduced.     

Effect of anisotropic reactivity on the rate of diffusion-controlled reactions: comparative analysis of the models of patches and hemispheres

A comparative analysis of two models of anisotropic reactivity in bimolecular diffusion-controlled reaction kinetics is presented. One is the conventional model of reactive patches (MRP), where the surface of a molecule is assumed to be reactive over a certain region (circular patch) with the rest of the surface being inert. Another one is the model of reactive hemispheres (MRH), assuming that a molecule is reactive within a certain distance from a point on its surface. The accuracy of the known and newly derived simple analytical expressions for the reaction rate is tested by comparison with the simulation results obtained by the original Brownian dynamics method. These formulas prove to be quite accurate in the practically important limit of strong anisotropy corresponding to small size of the reactive patches or hemispheres. Numerical calculations confirm earlier predictions that the MRP rates are much smaller than the MRH rates for the same radii of the reactive regions, especially in the case where both reacting molecules are anisotropic.     

Does random collisional cross-linking occur?

In fluid membranes, mobile molecules are thought to collide at high frequencies. Concern has been expressed as to whether these colliding molecules are cross-linked during the chemical cross-linking of membrane molecules, thereby creating problems in interpreting such experiments. Hemoglobin was used as a model to test this possibility. Oligomers larger than the tetramer could be cross-linked depending on factors such as hemoglobin concentration, duration of the cross-linking reaction and the type of reagent. Under certain conditions, however, such as a hemoglobin concentration less than 150 μM or a duration of cross-linking shorter than 15 min, larger oligomers were not detectable. Analysis of these data suggests that the probability of random collisional cross-links under normal conditions is insignificant.     

The analysis of stress-induced duplex destabilization in long genomic DNA sequences.

We present a method for calculating predicted locations and extents of stress-induced DNA duplex destabilization (SIDD) as functions of base sequence and stress level in long DNA molecules. The base pair denaturation energies are assigned individually, so the influences of near neighbors, methylated bases, adducts, or lesions can be included. Sample calculations indicate that copolymeric energetics give results that are close to those derived when full near-neighbor energetics are used; small but potentially informative differences occur only in the calculated SIDD properties of moderately destabilized regions. The method presented here for analyzing long sequences calculates the destabilization properties within windows of fixed length N, with successive windows displaced by an offset distance d(o). The final values of the relevant destabilization parameters for each base pair are calculated as weighted averages of the values computed for each window in which that base pair appears. This approach implicitly assumes that the strength of the direct coupling between remote base pairs that is induced by the imposed stress attenuates with their separation distance. This strategy enables calculations of the destabilization properties of DNA sequences of any length, up to and including complete chromosomes. We illustrate its utility by calculating the destabilization properties of the entire E. coli genomic DNA sequence. A preliminary analysis of the results shows that promoters are associated with SIDD regions in a highly statistically significant manner, suggesting that SIDD attributes may prove useful in the computational prediction of promoter locations in prokaryotes.     

THE DARK ADAPTATION OF THE HUMAN EYE

During the dark adaptation of the human eye, its visual threshold decreases to a small fraction of its original value in the light. An analysis of the quantitative data describing this adaptation shows that it follows the course of a bimolecular chemical reaction. On the basis of these findings it is suggested that visual reception in dim light is conditioned by a reversible photochemical reaction involving a photosensitive substance and its two products of decomposition. Accordingly, dark adaptation depends on the course of the "dark" reaction during which the two products of decomposition reunite to synthesize the original photosensitive substance.     

Derivation of the Formula to Calculate the Chemical Potential Difference for the Components Involved in Identity Exchange Moves in Gibbs Ensemble Simulation of Mixtures

Since temperature and pressure are specified at the beginning of a simulation run in the Gibbs ensemble Monte Carlo (GEMC) method for mixtures, the condition of equilibrium is fulfilled through two sets of equalities in each phase: one set for the chemical potentials of the components of smaller molecules (calculated from transfer trial moves), and the other set for the differences between the chemical potentials of the components of larger molecules and the components of smaller molecules (calculated from identity exchange trial moves). The formula to calculate the former quantities is known since the time the GEMC method was proposed. However, the formula to calculate the latter quantities has recently been given in the literature without a formal derivation. In this work, a statistical-mechanical derivation of that formula is presented, within the framework of the canonical ensemble, and some justification is given for its extension to the Gibbs ensemble.     

Structural determination of complex natural products by quantum mechanical calculations of <Superscript>13</Superscript>C NMR chemical shifts: development of a parameterized protocol for terpenes

Nuclear magnetic resonance (NMR) spectroscopy is one of the most important tools for determining the structures of organic molecules. Despite the advances made in this technique, revisions of erroneously established structures for natural products are still commonly published in the literature. In this context, the prediction of chemical shifts through ab initio and density functional theory (DFT) calculations has become a very powerful tool for assisting with the structural determination of complex organic molecules. In this work, we present the development of a protocol for ¹³C chemical shift calculations of terpenes, a class of natural products that are widely distributed among plant species and are very important due to their biological and pharmacological activities. This protocol consists of GIAO-DFT calculations of chemical shifts and the application of a parameterized scaling factor in order to ensure accurate structural determination of this class of natural products. The application of this protocol to a set of five terpenes yielded accurate calculated chemical shifts, showing that this is a very attractive tool for the calculation of complex organic structures such as terpenes.     

Multitrait fine mapping of quantitative trait loci using combined linkage disequilibria and linkage analysis

A novel multitrait fine-mapping method is presented. The method is implemented by a model that treats QTL effects as random variables. The covariance matrix of allelic effects is proportional to the IBD matrix, where each element is the probability that a pair of alleles is identical by descent, given marker information and QTL position. These probabilities are calculated on the basis of similarities of marker haplotypes of individuals of the first generation of genotyped individuals, using "gene dropping" (linkage disequilibrium) and transmission of markers from genotyped parents to genotyped offspring (linkage). A small simulation study based on a granddaughter design was carried out to illustrate that the method provides accurate estimates of QTL position. Results from the simulation also indicate that it is possible to distinguish between a model postulating one pleiotropic QTL affecting two traits vs. one postulating two closely linked loci, each affecting one of the traits.     

Microcalorimetric determination of thermochemical parameters of the phosphofructokinase reaction]

A calorimetric procedure for determining deltaH, deltaG, deltaS and Keq of a bimolecular reaction with two or more products is described. By using this method the thermodynamic parameters of the phosphofructokinase reaction are determined. At pH 7.0 and 25 degrees C a reaction enthalpy of-6.96kcal/mole was found after correction for the neutralization enthalpy of the buffer and of the enthalpy difference of the magnesium complexes of ATP and ADP, respectively. The free energy of the phosphofructokinase reaction has been found under these conditions to be -3.96kcal/mole.     

Kinetic and Energetic Model for the Primary Processes in Photosystem II

A detailed model for the kinetics and energetics of the exciton trapping, charge separation, charge recombination, and charge stabilization processes in photosystem (PS) II is presented. The rate constants describing these processes in open and closed reaction centers (RC) are calculated on the basis of picosecond data (Schatz, G. H., H. Brock, and A. R. Holzwarth. 1987. Proc. Natl. Acad. Sci. USA. 84:8414-8418) obtained for oxygen-evolving PS II particles from Synechococcus sp. with ~80 chlorophylls/P₆₈₀. The analysis gives the following results. (a) The PS II reaction center donor chlorophyll P₆₈₀ constitutes a shallow trap, and charge separation is overall trap limited. (b) The rate constant of charge separation drops by a factor of ~6 when going from open (Q-oxidized) to closed (Q-reduced) reaction centers. Thus the redox state of Q controls the yield of radical pair formation and the exciton lifetime in the Chl antenna. (c) The intrinsic rate constant of charge separation in open PS II reaction centers is calculated to be ~2.7 ps^-1. (d) In particles with open RC the charge separation step is exergonic with a decrease in standard free energy of ~38 meV. (e) In particles with closed RC the radical pair formation is endergonic by ~12 meV. We conclude on the basis of these results that the long-lived (nanoseconds) fluorescence generally observed with closed PS II reaction centers is prompt fluorescence and that the amount of primary radical pair formation is decreased significantly upon closing of the RC.     

Proton nuclear magnetic resonance investigations of fraying in double-stranded d-ApTpGpCpApT in H2O solution.

The chemical shifts and line widths of the Watson-Crick ring NH resonances of the self-complementary duplex of d-ApTpGpApT have been monitored at low ionic strength and in the presence of Mg ions at neutral pH in aqueous solution to determine the thermodynamic parameters associated with fraying (D. J. Patel (1974), Biochemistry 13, 2396) at the terminal and internal base pairs as a function of temperature and pH. From studies in H2O-MeOH (3:2), the fraying process persists down to approximately -20 degrees for the internal TA base pair and down to and probably beyond -30 degrees for the terminal AT base pair. The observed average chemical shift at each of these base pairs as a function of temperature suggests rapid exchange on the nuclear magnetic resonance (NMR) time scale between helix and coil (chemical shift separation of 3.2 ppm) and have been utilized to determine the dissociation constant at the terminal and internal base pairs. Comparison of the reaction enthalpies elucidated from the chemical shift parameters with those reported from optical studies suggests that the symmetry related internal TA base pairs break in a coupled manner at low ionic strength, with the coupling removed in the presence of Mg ions and high salt. By contrast, the symmetry related terminal AT base pairs break independently of each other in the absence and presence of Mg ions and high salt. The terminal base pair exhibits a Tm of 10-15 degrees lower than that of the internal base pair in the hexanucleotide, with divalent Mg ions and high salt stabilizing the double helix as reflected in the Tm values of these base pairs. The observed line width changes as a function of temperature provide an estimate of the exchange rate of the proton from the coil form with water. The exchange reaction from the coil state is base catalyzed with rate constants in the diffusion limit.     

Investigating protein-protein interaction surfaces using a reduced stereochemical and electrostatic model

A method of calculating the electrostatic potential energy between two molecules, using finite difference potential, is presented. A reduced charge set is used so that the interaction energy can be calculated as the two static molecules explore their full six-dimensional configurational space. The energies are contoured over surfaces fixed to each molecule with an interactive computer graphics program. For two crystal structures (trypsin-trypsin inhibitor and anti-lysozyme Fab-lysozyme), it is found that the complex corresponds to highly favourable interacting regions in the contour plots. These matches arise from a small number of protruding basic residues interacting with enhanced negative potential in each case. The redox pair cytochrome c peroxidase-cytochrome c exhibits an extensive favourably interacting surface within which a possible electron transfer complex may be defined by an increased electrostatic complementarity, but a decreased electrostatic energy. A possible substrate transfer configuration for the glycolytic enzyme pair glyceraldehyde phosphate dehydrogenase-phosphoglycerate kinase is presented.     

Phase diagrams for impure lipid systems. Application to lipid/anaesthetic mixtures

A physical model is presented to describe theoretically the temperature-dependent interactions of lipid bilayers with small molecules such as anaesthetics. Based on an earlier model, a triangular lattice in which each site is occupied by a single lipid chain is constructed and the small (anaesthetic) molecules are assumed to occupy interstitial sites in the centre of each lattice triangle. The phase characteristics of such lipid/anaesthetic mixtures are described in terms of the interaction parameters between lipid-lipid, lipid-anaesthetic and anaesthetic-anaesthetic molecules. Depending on the chemical nature of the interacting species the following three models are formulated: Model I. An interstitial model in which the only perturbation is in the head-group region of the bilayer and direct interactions between neighbouring anaesthetic molecules are taken into account. Model II. Here, only hydrophobic interactions between anaesthetics and lipids are considered. Model III. Both van der Waals' and coulombic interactions are taken into account. Phase diagrams for the three models are obtained by numerical calculation over a wide range of interaction parameters. It is shown that in all three models, lateral phase separation takes place due to the presence of anaesthetics. The heat of transition, however, is found to be virtually independent of the anaesthetic concentration.     

Higher-order complex formation between the 72-kilodalton type IV collagenase and tissue inhibitor of metalloproteinases-2.

The collagenases are a class of matrix degradative enzymes whose actions are important in physiological and pathological processes. The human 72-kDa type IV collagenase (matrix metalloproteinase-2) and its proteinase inhibitor, tissue inhibitor of metalloproteinases-2 (TIMP-2), are produced as a proenzyme-inhibitor complex by numerous cell lines. We analyzed the quaternary structure of and enzyme-inhibitor interactions in the native enzyme-inhibitor complex by studying the pattern of complexes demonstrated by molecular weight determination in nondenaturing polyacrylamide gels and evaluating the products formed by reaction of the native complexes with cross-linking agents. Electrophoresis in native polyacrylamide gels demonstrates that approximately 79% of the latent enzyme is present in a 1:1 bimolecular complex with the inhibitor TIMP-2, with 21% present as a complete tetrameric complex of two molecules of collagenase combined with two molecules of TIMP-2. The enzyme complex activated with organomercurials displays a shift to a higher proportion of the bimolecular complex with only 5% present as higher molecular weight complexes. Cross-linking of the latent and active forms of the complex with bis(sulfosuccinimidyl) suberate (BS3) and bis(sulfosuccinimidyl) tartarate demonstrates both the 1:1 and 2:2 complexes as well as an intermediate form that appears to be a complex composed of two molecules of collagenase and one of TIMP-2. The distribution of cross-linked products is unchanged with the addition of excess TIMP-2 to the reaction mix, implying that the binding sites for TIMP-2 to the initial enzyme-inhibitor complex are all occupied when the stoichiometry is 1 to 1.(ABSTRACT TRUNCATED AT 250 WORDS)     

CAST AWAY, a membrane-associated receptor-like kinase, inhibits organ abscission in Arabidopsis

Receptor-like kinase-mediated cell signaling pathways play fundamental roles in many aspects of plant growth and development. A pair of Arabidopsis (Arabidopsis thaliana) leucine-rich repeat receptor-like kinases (LRR-RLKs), HAESA (HAE) and HAESA-LIKE2 (HSL2), have been shown to activate the cell separation process that leads to organ abscission. Another pair of LRR-RLKs, EVERSHED (EVR) and SOMATIC EMBRYOGENESIS RECEPTOR-LIKE KINASE1, act as inhibitors of abscission, potentially by modulating HAE/HSL2 activity. Cycling of these RLKs to and from the cell surface may be regulated by NEVERSHED (NEV), a membrane trafficking regulator that is essential for organ abscission. We report here the characterization of CAST AWAY (CST), a receptor-like cytoplasmic kinase that acts as a spatial inhibitor of cell separation. Disruption of CST suppresses the abscission defects of nev mutant flowers and restores the discrete identity of the trans-Golgi network in nev abscission zones. After organ shedding, enlarged abscission zones with obscured boundaries are found in nev cst flowers. We show that CST is a dual-specificity kinase in vitro and that myristoylation at its amino terminus promotes association with the plasma membrane. Using the bimolecular fluorescence complementation assay, we have detected interactions of CST with HAE and EVR at the plasma membrane of Arabidopsis protoplasts and hypothesize that CST negatively regulates cell separation signaling directly and indirectly. A model integrating the potential roles of receptor-like kinase signaling and membrane trafficking during organ separation is presented.     

Multi-scale stochastic simulation of diffusion-coupled agents and its application to cell culture simulation

Many biological systems consist of multiple cells that interact by secretion and binding of diffusing molecules, thus coordinating responses across cells. Techniques for simulating systems coupling extracellular and intracellular processes are very limited. Here we present an efficient method to stochastically simulate diffusion processes, which at the same time allows synchronization between internal and external cellular conditions through a modification of Gillespie's chemical reaction algorithm. Individual cells are simulated as independent agents, and each cell accurately reacts to changes in its local environment affected by diffusing molecules. Such a simulation provides time-scale separation between the intra-cellular and extra-cellular processes. We use our methodology to study how human monocyte-derived dendritic cells alert neighboring cells about viral infection using diffusing interferon molecules. A subpopulation of the infected cells reacts early to the infection and secretes interferon into the extra-cellular medium, which helps activate other cells. Findings predicted by our simulation and confirmed by experimental results suggest that the early activation is largely independent of the fraction of infected cells and is thus both sensitive and robust. The concordance with the experimental results supports the value of our method for overcoming the challenges of accurately simulating multiscale biological signaling systems.