Mechano-Chemical Coupling at Molecular Level: Forced Dissociation of Selectin/Ligand Binding

Mechano-chemical coupling is a common phenomenon that exists in various biological processes at different physiological levels. Bone tissue remodeling strongly depends on the local mechanical load. Leukocytes are sheared to form the transient aggregates with platelets or other leukocytes in the circulation. Flow pattern affects the signal transduction pathways in endothelial cells. Receptor/ligand interactions are important to cell adhesion since they supply the physical linkages...

Forced Dissociation of Selectin-ligand Complexes Using Steered Molecular Dynamics Simulation

Selectin-ligand interactions are crucial to such biological processes as inflammatory cascade or tumor metastasis. How transient formation and dissociation of selectin-ligand bonds in blood flow are coupled to molecular conformation at atomic level, however, has not been well understood. In this study, steered molecular dynamics (SMD) simulations were used to elucidate the intramolecular and intermolecular conformational evolutions involved in forced dissociation of three selectin-ligand systems: the construct consisting of P-selectin lectin (Lec) and epidermal growth factor (EGF)-like domains (P-LE) interacting with synthesized sulfoglycopeptide or SGP-3, P-LE with sialyl Lewis X (sLeX), and E-LE with sLeX. SMD simulations were based on newly built-up force field parameters including carbohydrate units and sulfated tyrosine(s) using an analogy approach. The simulations demonstrated that the complex dissociation was coupled to the molecular extension. While the intramolecular unraveling in P-LESGP-3 system mainly resulted from the destroy of the two anti-parallel sheets of EGF domain and the breakage of hydrogen-bond cluster at the Lec-EGF interface, the intermolecular dissociation was mainly determined by separation of fucose (FUC) from Ca2+ ion in all three systems. Conformational changes during forced dissociations depended on pulling velocities and forces, as well as on how the force was applied. This work provides an insight into better understanding of conformational changes and adhesive functionality of selectin-ligand interactions under external forces.

Optical Factors Determined by the T-matrix Method in Turbidity Measurement of Absolute Coagulation Rate Constants

Turbidity measurement for the absolute coagulation rate constant of suspensions has been extensively adopted because of its simplicity and easy implementation. A key factor to derive the rate constant from experimental data is how to theoretically evaluate the so-called optical factor involved in calculating the extinction cross section of doublets formed in the aggregation. In a previous paper, we have shown that compared with other theoretical approaches, the T-matrix method provides a robust solution to this problem and is effective in extending the applicability range of the turbidity methodology as well as increasing measurement accuracy. This paper will provide a more comprehensive discussion about the physical insight of using the T-matrix method in turbidity measurement and associated technical details. In particular, the importance of ensuring the correct value for the refractive indices for colloidal particles and the surrounding medium used in the calculation is addressed because the indices generally vary with the wavelength of the incident light. The comparison of calculated results with experiments shows that the T-matrix method can correctly calculate optical factors even for large particles, whereas other existing theories cannot. In addition, the calculated data of the optical factor by the T-matrix method for a range of particle radii and incident light wavelengths are listed.

Toward an understanding of the turbidity measurement of fleterocoagulation rate constants of dispersions containing particles of different sizes

Our previous studies have shown that the determination of coagulation rate constants by turbidity measurement becomes impossible for a certain operating wavelength (that is, its blind point) because at this wavelength the change in the turbidity of a dispersion completely loses its response to the coagulation process. Therefore, performing the turbidity measurement in the wavelength range near the blind point should be avoided. In this article, we demonstrate that the turbidity measurement of the rate constant for coagulation of a binary dispersion containing particles of two different sizes (heterocoagulation) presents special difficulties because the blind point shifts with not only particle size but also with the component fraction. Some important aspects of the turbidity measurement for the heterocoagulation rate constant are discussed and experimentally tested. It is emphasized that the T-matrix method can be used to correctly evaluate extinction cross sections of doublets formed during the heterocoagulation process, which is the key data determining the rate constant from the turbidity measurement, and choosing the appropriate operating wavelength and component fraction are important to achieving a more accurate rate constant. Finally, a simple scheme in experimentally determining the sensitivity of the turbidity changes with coagulation over a wavelength range is proposed.

A new expression of hardening coefficients for fcc-crystal and calibration of the material constants

In order to describe the effect of latent hardening on the macro-plastic behavior of foc-crystal, a new expression for hardening coefficient is proposed in which there are 12 material constants, each having clear physical meaning. And a method of material constant calibration is suggested and used to determine the material constants of copper and aluminum crystal. The simulated load-elongation curves along various crystallographic orientations are comparable with the experimental ones.

2D Kinetics and Forced Dissociation of Selectin-ligand Bindings

Cell adhesion is crucial to many pathophysiological processes, such as inflammatory reaction and tumor metastasis. It is mediated by specific interactions between receptors and ligands, and provides the physical linkages among cells. For example, interactions between selectins and glycoconjugate ligands mediate leukocyte initially tethering to and subsequently rolling on vascular surfaces in sites of inflammation or injury, which is determined by their fast kinetic rates. To mediate cell adhesion, the interacting receptors and ligands must anchor to apposing surfaces of two cells or a cell and the substratum, i.e. , the so-called two-dimensional (2D) binding, which differs from interactions in the fluid phase, i.e. , the three-dimensional (3D) binding. How structural variations and surface environments of interacting molecules affect their 2D kinetics, and how external forces manipulate their dissociation has little been known quantitatively, and nowadays attracts more and more attentions.

Mechanisms underlying two kinds of surface effects on elastic constants

ABSTRACT Recently, people are confused with two opposite variations of elastic modulus with decreasing size of nano scale sample: elastic modulus either decreases or increases with decreas- ing sample size. In this paper, based on intermolecular potentials and a one dimensional model, we provide a unified understanding of the two opposite size effects. Firstly, we analyzed the mi- crostructural variation near the surface of an fcc nanofilm based on the Lennard-Jones potential. It is found that the atomic lattice near the surface becomes looser in comparison with the bulk, indicating that atoms in the bulk are located at the balance of repulsive forces, resulting in the decrease of the elastic moduli with the decreasing thickness of the film accordingly. In addition, the decrease in moduli should be attributed to both the looser surface layer and smaller coor- dination number of surface atoms. Furthermore, it is found that both looser and tighter lattice near the surface can appear for a general pair potential and the governing mechanism should be attributed to the surplus of the nearest force to all other long range interactions in the pair po- tential. Surprisingly, the surplus can be simply expressed by a sum of the long range interactions and the sum being positive or negative determines the looser or tighter lattice near surface re- spectively. To justify this concept, we examined ZnO in terms of Buckingham potential with long range Coulomb interactions. It is found that compared to its bulk lattice, the ZnO lattice near the surface becomes tighter, indicating the atoms in the bulk located at the balance of attractive forces, owing to the long range Coulomb interaction. Correspondingly, the elastic modulus of one- dimensional ZnO chain increases with decreasing size. Finally, a kind of many-body potential for Cu was examined. In this case, the surface layer becomes tighter than the bulk and the modulus increases with deceasing size, owing to the long range repulsive pair interaction, as well as the cohesive many-body interaction caused by the electron redistribution.

Investigation of competitive antagonist binding to the nicotinic acetylcholine receptor using voltage-jump and light-flash techniques

1. The effect of 2,2’-bis-[α-(trimethylammonium)methyl]azobenzene (2BQ), a photoisomerizable competitive antagonist, was studied at the nicotinic acetycholine receptor of Electrophorus electroplaques using voltage-jump and light-flash techniques.

2. 2BQ, at concentrations below 3 μΜ, reduced the amplitude of voltage-jump relaxations but had little effect on the voltage-jump relaxation time constants under all experimental conditions. At higher concentrations and voltages more negative than -150 mV, 2BQ caused significant open channel blockade.

3. Dose-ratio studies showed that the cis and trans isomers of 2BQ have equilibrium binding constants (K _ᵢ) of .33 and 1.0 μΜ, respectively. The binding constants determined for both isomers are independent of temperature, voltage, agonist concentration, and the nature of the agonist.

4. In a solution of predominantly cis-2BQ, visible-light flashes led to a net cis→trans isomerization and caused an increase in the agonist-induced current. This increase had at least two exponential components; the larger amplitude component had the same time constant as a subsequent voltage-jump relaxation; the smaller amplitude component was investigated using ultraviolet light flashes.

5. In a solution of predominantly trans-2BQ, UV-light flashes led to a net trans→cis isomerization and caused a net decrease in the agonist-induced current. This effect had at least two exponential components. The smaller and faster component was an increase in agonist-induced current and had a similar time constant to the voltage-jump relaxation. The larger component was a slow decrease in the agonist-induced current with rate constant approximately an order of magnitude less than that of the voltage-jump relaxation. This slow component provided a measure of the rate constant for dissociation of cis-2BQ (k_ = 60/s at 20°C). Simple modelling of the slope of the dose-rate curves yields an association rate constant of 1.6 x 10⁸/M/s. This agrees with the association rate constant of 1.8 x 10⁸/M/s estimated from the binding constant (K_i). The Q₁₀ of the dissociation rate constant of cis-2BQ was 3.3 between 6° and 20°C. The rate constants for association and dissociation of cis-28Q at receptors are independent of voltage, agonist concentration, and the nature of the agonist.

6. We have measured the molecular rate constants of a competitive antagonist which has roughly the same K _ᵢ as d-tubocurarine but interacts more slowly with the receptor. This leads to the conclusion that curare itself has an association rate constant of 4 x 10⁹/M/s or roughly as fast as possible for an encounter-limited reaction.

I. Electron spin relaxation studies of manganese(II) complexes in acetonitrile. II. Nuclear spin relaxation studies of bromine in tetrabromomanganese(II) in acetonitrile

Magnetic resonance techniques have given us a powerful means for investigating dynamical processes in gases, liquids and solids. Dynamical effects manifest themselves in both resonance line shifts and linewidths, and, accordingly, require detailed analyses to extract desired information. The success of a magnetic resonance experiment depends critically on relaxation mechanisms to maintain thermal equilibrium between spin states. Consequently, there must be an interaction between the excited spin states and their immediate molecular environment which promote changes in spin orientation while excess magnetic energy is coupled into other degrees of freedom by non-radiative processes. This is well known as spin-lattice relaxation. Certain dynamical processes cause fluctuations in the spin state energy levels leading to spin-spin relaxation and, here again, the environment at the molecular level plays a significant role in the magnitude of interaction. Relatively few electron spin relaxation studies of solutions have been conducted and the present work is addressed toward the extension of our knowledge in this area and the retrieval of dynamical information from line shape analyses on a time scale comparable to diffusion controlled phenomena.

Specifically, the electron spin relaxation of three Mn⁺²3d⁵ complexes, Mn(CH₃CN)₆⁺², MnCl₄^-2 in acetonitrile has been studied in considerable detail. The effective spin Hamiltonian constants were carefully evaluated under a wide range of experimental conditions. Resonance widths of these Mn⁺² complexes were studied in the presence of various excess ligand ions and as a function of concentration, viscosity, temperature and frequency (X-band, ~9.5 Ԍ Hz and K-band, ~35 Ԍ Hz).

A number of interesting conclusions were drawn from these studies. For the Et₄NCl-₄^-2 system several relaxation mechanisms leading to resonance broadening were observed. One source appears to arise through spin-orbit interactions caused by modulation of the ligand field resulting from transient distortions of the complex imparted by solvent fluctuations in the immediate surroundings of the paramagnetic ion. An additional spin relaxation was assigned to the formation of ion pairs [Et₄N⁺…MnCl₄^-2] and it was possible to estimate the dissociation constant for this specie in acetonitrile.

The Bu₄NBr-MnBr₄^-2 study was considerably more interesting. As in the former case, solvent fluctuations and ion-pairing of the paramagnetic complex [Bu₄N⁺…MnBr₄^-2] provide significant relaxation for the electronic spin system. Most interesting, without doubt, is the onset of a new relaxation mechanism leading to resonance broadening which is best interpreted as chemical exchange. Thus, assuming that resonance widths were simply governed by electron spin state lifetimes, we were able to extract dynamical information from an interaction in which the initial and final states are the same

MnBr₄^-2 + Br^- = MnBr₄^-2 + Br^-.

The bimolecular rate constants were obtained at six different temperatures and their magnitudes suggested that the exchange is probably diffusion controlled with essentially a zero energy of activation. The most important source of spin relaxation in this system stems directly from dipolar interactions between the manganese 3d⁵ electrons. Moreover, the dipolar broadening is strongly frequency dependent indicating a deviation between the transverse and longitudinal relaxation times. We are led to the conclusion that the 3d⁵ spin states of ion-paired MnBr₄^-2 are significantly correlated so that dynamical processes are also entering the picture. It was possible to estimate the correlation time, ^Td, characterizing this dynamical process.

In Part II we study nuclear magnetic relaxation of bromine ions in the MnBr4-2-Bu4NBr-acetonitrile system. Essentially we monitor the 79Br and 81Br linewidths in response to the [MnBr4-2]/[Br-] ratio with the express purpose of supporting our contention that exchange is occurring between "free" bromine ions in the solvent and bromine in the first coordination sphere of the paramagnetic anion. The complexity of the system elicited a two-part study: (1) the linewidth behavior of Bu4NBr in anhydrous CH3CN in the absence of MnBr4-2 and (2) in the presence of MnBr4-2. It was concluded in study (1) that dynamical association, Bu4NBr k1= Bu4N+ + Br-, was modulating field-gradient interactions at frequencies high enough to provide an estimation of the unimolecular rate constant, k1. A comparison of the two isotopic bromine linewidth-mole fraction results led to the conclusion that quadrupole interactions provided the dominant relaxation mechanism. In study (2) the "residual" bromine linewidths for both 79Br and 81Br are clearly controlled by quadrupole interactions which appear to be modulated by very rapid dynamical processes other than molecular reorientation. We conclude that the "residual" linewidth has its origin in chemical exchange and that bromine nuclei exchange rapidly between a "free" solvated ion and the paramagnetic complex, MnBr4-2.

Part I. Electric birefringence studies of deoxyribonucleic acids. Part II. Selective dissociation of nucleohistone complexes

Part I

The electric birefringence of dilute DNA solutions has been studied in considerable detail and on a large number of samples, but no new and reliable information was discovered concerning the tertiary structure of DNA. The large number of variables which effect the birefringence results is discussed and suggestions are made for further work on the subject.

The DNA molecules have been aligned in a rapidly alternating (10 to 20 kc/sec) square wave field confirming that the orientation mechanism is that of counterion polarization. A simple empirical relation between the steady state birefringence, Δn_st, and the square of the electric field, E, has been found: Δn_st = E²/(a E² + b), where a = 1/Δn_s and b = (E²/Δn_st)_E→o. Δn_s is the birefringence extrapolated to infinite field strength.

The molecules show a distribution of relaxation times from 10^-4 to 0.2 sec, which is consistent with expectations for flexible coil molecules. The birefringence and the relaxation times decrease with increasing salt concentrations. They also depend on the field strength and pulse duration in a rather non-reproducible manner, which may be due in part to changes in the composition of the solution or in the molecular structure of the DNA (other than denaturation). Further progress depends on the development of some control over these effects.

Part II

The specificity of the dissociation of reconstituted and native deoxyribonucleohistones (DNH) by monovalent salt solutions has been investigated. A novel zone ultracentrifugation method is used in which the DNH is sedimented as a zone through a preformed salt gradient, superimposed on a stabilizing D₂O (sucrose) density gradient. The results, obtained by scanning the quartz sedimentation tubes in a spectrophotometer, were verified by the conventional, preparative sedimentation technique. Procedures are discussed for the detection of microgram quantities of histones, since low concentrations must be used to prevent excessive aggregation of the DNH.

The data show that major histone fractions are selectively dissociated from DNH by increasing salt concentrations: Lysine rich histone (H I) dissociates gradually between 0.1 and 0.3 F, slightly lysine rich histone (H II) dissociates as a narrow band between 0.35 and 0.5 F, and arginine rich histone (H III, H IV) dissociates gradually above 0.5 F NaClO₄.

The activity of the partially dissociated, native DNH in sustaining RNA synthesis, their mobility and their unusual heat denaturation and renaturation behavior are described. The two-step melting behavior of the material indicates that the histones are non-randomly distributed along the DNA, but the implications are that the uncovered regions are not of gene-size length.

Nature of the radical intermediates in a substituted cyclopropylcarbiny-allylcarbinyl system

We have sought to determine the nature of the free-radical precursors to ring-opened hydrocarbon 5 and ring-closed hydrocarbon 6. Reasonable alternative formulations involve the postulation of hydrogen abstraction (a) by a pair of rapidly equilibrating classical radicals (the ring-opened allylcarbinyl-type radical 3 and the ring-closed cyclopropylcarbinyl-type 4), or (b) by a nonclassical radical such as homoallylic radical 7.

[Figure not reproduced.]

Entry to the radical system is gained via degassed thermal decomposition of peresters having the ring-opened and the ring-closed structures. The ratio of 6:5 is essentially independent of the hydrogen donor concentration for decomposition of the former at 125° in the presence of triethyltin hydrdride. A deuterium labeling study showed that the α and β methylene groups in 3 (or the equivalent) are rapidly interchanged under these conditions.

Existence of two (or more) product-forming intermediates is indicated (a) by dependence of the ratio 6:5 on the tin hydride concentration for decomposition of the ring-closed perester at 10 and 35°, and (b) by formation of cage products having largely or wholly the structure (ring-opened or ring-closed) of the starting perester.

Relative rates of hydrogen abstraction by 3 could be inferred by comparison of ratios of rate constants for hydrogen abstraction and ortho-ring cyclization:

[Figure not reproduced.]

At 100° values of k_a/k_r are 0.14 for hydrogen abstraction from 1,4-cyclohexadiene and 7 for abstraction from triethyltin hydride. The ratio 6:5 at the same temperature is ~0.0035 for hydrogen abstraction from 1,4-cyclohexadiene, ~0.078 for abstraction from the tin hydride, and ≥ 5 for abstraction from cyclohexadienyl radicals. These data indicate that abstraction of hydrogen from triethyltin hydride is more rapid than from 1,4-cyclohexadiene by a factor of ~1000 for 4, but only ~50 for 3.

Measurements of product ratios at several temperatures allowed the construction of an approximate energy-level scheme. A major inference is that isomerization of 3 to 4 is exothermic by 8 ± 3 kcal/mole, in good agreement with expectations based on bond dissociation energies. Absolute rate-constant estimates are also given.

The results are nicely compatible with a classical-radical mechanism, but attempted interpretation in terms of a nonclassical radical precursor of product ratios formed even from equilibrated radical intermediates leads, it is argued, to serious difficulties.

The roles played by hydrogen abstraction from 1,4,-cyclohexadiene and from the derived cyclohexadienyl radicals were probed by fitting observed ratios of 6:5 and 5:10 in the sense of least-squares to expressions derived for a complex mechanistic scheme. Some 30 to 40 measurements on each product ratio, obtained under a variety of experimental conditions, could be fit with an average deviation of ~6%. Significant systematic deviations were found, but these could largely be redressed by assuming (a) that the rate constant for reaction of 4 with cyclohexadienyl radical is inversely proportional to the viscosity of the medium (i.e., is diffusion-controlled), and (b) that k_a/k_r for hydrogen abstraction from 1,4-cyclohexadiene depends slightly on the composition of the medium. An average deviation of 4.4% was thereby attained.

Degassed thermal decomposition of the ring-opened perester in the presence of the triethyltin hydride occurs primarily by attack on perester of triethyltin radicals, presumably at the –O-O- bond, even at 0.01 M tin hydride at 100 and 125°. Tin ester and tin ether are apparently formed in closely similar amounts under these conditions, but the tin ester predominates at room temperature in the companion air-induced decomposition, indicating that attack on perester to give the tin ether requires an activation energy approximately 5 kcal/mole in excess of that for the formation of tin ester.

I. Non-linear effects in a self-consistent calculation of SU_3 symmetry breaking in strong interaction coupling constants. II. Direct-channel reggeization of strong interaction scattering amplitudes

The thesis is divided into two parts. Part I generalizes a self-consistent calculation of residue shifts from SU₃ symmetry, originally performed by Dashen, Dothan, Frautschi, and Sharp, to include the effects of non-linear terms. Residue factorizability is used to transform an overdetermined set of equations into a variational problem, which is designed to take advantage of the redundancy of the mathematical system. The solution of this problem automatically satisfies the requirement of factorizability and comes close to satisfying all the original equations.

Part II investigates some consequences of direct channel Regge poles and treats the problem of relating Reggeized partial wave expansions made in different reaction channels. An analytic method is introduced which can be used to determine the crossed-channel discontinuity for a large class of direct-channel Regge representations, and this method is applied to some specific representations.

It is demonstrated that the multi-sheeted analytic structure of the Regge trajectory function can be used to resolve apparent difficulties arising from infinitely rising Regge trajectories. Also discussed are the implications of large collections of "daughter trajectories."

Two things are of particular interest: first, the threshold behavior in direct and crossed channels; second, the potentialities of Reggeized representations for us in self-consistent calculations. A new representation is introduced which surpasses previous formulations in these two areas, automatically satisfying direct-channel threshold constraints while being capable of reproducing a reasonable crossed channel discontinuity. A scalar model is investigated for low energies, and a relation is obtained between the mass of the lowest bound state and the slope of the Regge trajectory.

Determination and analysis of the optical constants of thin films of nickel(II) and copper(II) hydrazone complexes by spectroscopic ellipsometry

Thin films of four nickel(II) and copper(II) hydrazone complexes, which will hopefully be used as recording layers for the next-generation of high-density recordable disks, were prepared by using the spin-coating method. Absorption spectra of the thin films on K9 optical glass substrates in the 300-700 nm wavelength region were measured. Optical constants (complex refractive indices N) and thickness d of the thin films prepared on single-crystal silicon substrates in the 275-675 nm wavelength region were investigated on a rotating analyzer-polarizer scanning ellipsometer by fitting the measured ellipsometric angles (Psi(lambda) and Delta(lambda)) with a 3-layer model (Si/dye film/air). The dielectric functions epsilon and absorption coefficients alpha as a function of the wavelength were then calculated. Additionally, a design to achieve high reflectivity and optimum dye film thickness with an appropriate reflective layer was performed with the Film Wizard software using a multilayered model (PC substrate/reflective layer/dye film/air) at 405 nm wavelength.

Calculations of bond dissociation energies and dipole moments in energetic materials using density-functional methods

We compare the effectiveness of six exchange/correlation functional combinations (Becke/Lee, Yang and Parr; Becke-3/Lee, Yang and Parr; Becke/Perdew-Wang 91; Becke-3/Perdew-Wang 91; Becke/Perdew 86; Becke-3/Perdew 86) for computing C-N, O-O and N-NO2 dissociation energies and dipole moments of five compounds. The studied compounds are hexabydro-1,3,5-trinitro-1,3,5-triazine (RDX), dimethylnitramine, cyanogen, nitromethane and ozone. The Becke-3/Perdew 86 in conjunction with 6-31G