Dielectric behavior of DNA in water–organic co-solvent mixtures

The radiowave dielectric dispersions of DNA in different water–organic co-solvent mixtures have been measured in the frequency range from 100 kHz to 100 MHz, where the polarization mechanism is generally attributed to the confinement of counterions within some specific lengths, either along tangential or perpendicular to the polyion chain. The dielectric dispersions have been analyzed on the basis of two partially different dielectric models, a continuum counterion fluctuation model proposed by Mandel and a discrete charged site model, proposed by Minakata. The influence of the quality of the solvent on the dielectric parameters has been investigated in water–methanol and water–glycerol mixtures at different composition, by varying the permittivity ?_m and the viscosity η of the solvent phase. The analysis of the dielectric spectra in solvents where electrostatic and hydrodynamic interactions vary with the solvent composition suggests that both the two models are able, in principle, to account for the observed high-frequency dielectric behavior. However, while some certain assumptions are necessary about the polyion structure within the Mandel model, no structural prerequisite is needed within the Minakata model, where the polarization mechanism invoked considers a radial counterion exchange with the outer medium, which is largely independent of the local polyion conformation.     

The delta-crystallin enhancer-binding protein delta EF1 is a repressor of E2-box-mediated gene activation.

The repressor delta EF1 was discovered by its action on the DC5 fragment of the lens-specific delta 1-crystallin enhancer. C-proximal zinc fingers of delta EF1 were found responsible for binding to the DC5 fragment and had specificity to CACCT as revealed by selection of high-affinity binding sequences from a random oligonucleotide pool. CACCT is present not only in DC5 but also in the E2 box (CACCTG) elements which are the binding sites of various basic helix-loop-helix activators and also the target of an unidentified repressor, raising the possibility that delta EF1 accounts for the E2 box repressor activity. delta EF1 competed with E47 for binding to an E2 box sequence in vitro. In lymphoid cells, endogenous delta EF1 activity as a repressor was detectable, and exogenous delta EF1 repressed immunoglobulin kappa enhancer by binding to the kappa E2 site. Moreover, delta EF1 repressed MyoD-dependent activation of the muscle creatine kinase enhancer and MyoD-induced myogenesis of 10T1/2 cells. Thus, delta EF1 counteracts basic helix-loop-helix activators through binding site competition and fulfills the conditions of the E2 box repressor. In embryonic tissues, the most prominent site of delta EF1 expression is the myotome. Myotomal expression as well as the above results argues for a significant contribution of delta EF1 in regulation of embryonic myogenesis through the modulation of the actions of MyoD family proteins.     

Effect of counterion concentration on the dielectric behavior of a polypeptidic chain in the helix–coil transition

On the basis of the two-state model of a polyelectrolyte solution, the ion concentration in the polymer domain has been calculated by using the spherical Poisson–Boltzmann equation. The ion accumulation in the neighboring of the polyion influences, on different time scales, various electrical properties of the solution, in particular the low-frequency electrical conductivity and the high-frequency dielectric dispersion. These predictions have been compared with recent dielectric measurements on poly (L -glutamic acid) aqueous solutions during the conformational transition from the α-helix to random coil, and a satisfactory agreement has been found. This finding suggests that counterion distribution plays a different role in determining the electrical properties of charged polymer solutions, causing a electrophoretic contribution of the polymer domain to the electrical conductivity and influencing the high-frequency dielectric dispersion. © 1993 John Wiley & Sons, Inc.     

Monte-Carlo-self consistent field method in the polyelectrolyte theory

A new time saving numerical method for calculation of equilibrium potential and density distribution of mobile ions around the polyion in a polyelectrolyte system is proposed: the region around the polyion is being divided into two zones-internal and external; in the internal zone all the ions are accounted explicity with the aid of Monte-Carlo procedure; in the external zone the combined Monte-Carlo-self consistent field method proposed earlier is applied, an exchange of ions between regions is being implied. For 1:1 electrolyte the optimal choice of the boundary between the zones has been demonstrated. As an example of a more complicated system calculation for 2:2:1:1 electrolyte was carried out.     

The dielectric increments of aqueous polyelectrolyte solutions: a scaling approach

This paper deals with dielectric dispersion curves (covering a frequency range from a few Hz to 100 MHz) of Na-poly(styrene-sulfonate) of 65,000 < or = Mw < or = 1,060,000 g mol(-1) in aqueous solutions. The values of the low frequency (dielectric increment1) and high frequency (dielectric increment2) dielectric increments, obtained from the experimental curves matched to a superposition of two Cole-Cole equations, have been analyzed in terms of their concentration and molar mass dependence. The concentrations C (g l(-1)) of the various solutions were mostly situated in the transition regime defined by Odijk [T. Odijk, Macromolecules 12 (1979) 688] between the dilute regime (C < Cg*) and the semi-dilute one (C > C**), and wherein the characteristic concentration C* marks the onset of flexibility effects on the polyion behavior. It has been shown that in the concentration range Cg* < C < C** the increments in both frequency domains satisfy a scaling relation dielectric increment(j) = Bj M(nu j) (C/C*)(mu j) with molar mass independent exponents nu j and mu j changing around C*. Their values are different for dielectric increment1 and dielectric increment2, except for mu above C* where both increments appear to become concentration-independent. Below Cg*, in the dilute regime, the two dispersion domains seem to merge. The increment dielectric increment = relative permittivity (0) - high frequency limit of relative permittivity is molar mass independent if scaled to (C/Cg*). The molar mass dependence of the increments as a function of the macromolecular concentration rhoP, dielectric increment or dielectric increment(j) approximately Mgamma (rhoP)mu, also reveals differences between the different concentration regimes. Extrapolation from above Cg* to zero concentration is thus unjustified.     

Quasielastic light scattering by biopolymers. VI. Diffusion of mononucleosomes and oligonucleosomes in the presence of static and sinusoidal electric fields

Quasielastic light scattering and electrophoretic light scattering studies were carried out on mononucleosome and oligonucleosome systems. The electrophoretic light scattering experiments employed static and sinusoidal electric fields. Data are presented that suggest at least two relaxation modes. It is proposed that the small amplitude sinusoidal field effectively polarizes the ion atmosphere about the polyion, thus leading to an induced dipole moment that varies sinusoidally in time. This model is, in essence, an extension of the current interpretation of low-frequency dielectric dispersion data on DNA as being due to fluctuations of counterions along the polyion.     

Monte-Carlo - Self Consistent Field Method in the Polyelectrolyte Theory

Abstract

A new time saving numerical method for calculation of equilibrium potential and density distribution of mobile ions around the polyion in a polyelectrolyte system is proposed: the region around the polyion is being divided into two zones - internal and external; in the internal zone all the ions are accounted explicity with the aid of Monte-Carlo procedure; in the external zone the combined Monte-Carlo - self consistent field method proposed earlier is applied, an exchange of ions between regions is being implied. For 1:1 electrolyte the optimal choice of the boundary between the zones has been demonstrated. As an example of a more complicated system calculation for 2:2:1:1 electrolyte was carried out.     

Changes in the electrical and visco-elastic properties of bilayer lipid membranes during interaction with proteins and lipoproteins

A method for simultaneous registration of planar bilayer lipid membrane (BLM) DC conductance G, capacitance C, surface potential difference delta phi and transversal elasticity module E is developed. C, delta phi and E are proportional to the amplitude of the first, second and third harmonics of capacitance current respectively. A comparative study of the interaction of BLM with very low density lipoproteins (VLDL), influenza virus matrix protein (M-protein) and yeast invertase was carried out. The kinetics of delta phi, E and G changes at different concentrations of VLDL, and dependence of delta phi and G on M-protein and invertase concentration was investigated. It is shown for VLDL invertase and M-protein that the changes in delta phi and E occur before the change in G. The method used permits to study peculiarities of individual stages of interaction between charge particles, supramolecular structures and lipid membranes.     

A simple procedure for purifying mammalian duodenal Ca2+-binding proteins on a 100 mg scale and an investigation of the stoichiometry of their high-affinity binding of Ca2+ ions.

The capacity of E. coli cells to regulate intracellular pH (pHi) during net potassium uptake has been investigated. The data show: (a) that cells sense their intracellular pH; (b) that the pH gradient (delta pH) exerts a feedback regulation on pHi; (c) that a mechanism of regulation of pHi exists which may be independent of Na+ [Zilberstein, Agmon, Schuldiner & Padan (1982) J. Biol. Chem. 257, 3687-3691]; and (d) that cells have a limited capacity to raise their intracellular pH in the absence of net K+ transport.     

Electric behaviour of natural and demineralized bones. Dielectric properties up to 1 GHz

In this paper we have measured the dielectric spectrum of water-saturated bones in native and demineralized states up to 1 GHz in the time domain. A novel method of analysis of the time domain spectroscopy data has been used. The results show a dielectric dispersion centered around 400 MHz for native samples and around 200 MHz for demineralized ones. The proposed mechanism for this dispersion is the movement of polar side chains, which is in agreement with what happens in hydrated collagen fibres.     

肠毒素源性定居因子CS6在减素伤寒沙门氏菌中表达及免疫原性的研究

将编码肠毒素源性大肠杆菌定居因子抗原ＣＳ６基因克隆到ｐＸＬ６７０,转化ａｓｄ基因突变的Ｅ．ｃｏｌｉ Ｘ６０９７,获得重组质粒ｐＳＳ６４,再将后者转化至减毒的△ａｒｏＡ、△ａｒｏＣ、△ａｓｄ伤寒沙门氏菌,构建了无药物抗性且稳定的大肠杆菌和伤寒双价菌苗候选株。小鼠腹腔免疫和攻击实验表明,该菌株对伤寒沙门氏菌毒株的攻击具有良好的保护作用。家兔免疫实验证明,该菌株能产生抗ＣＳ６和伤寒菌Ｖｉ抗原的血清抗体。     

Gamma delta T cell-deficient mice have a down-regulated CD8+ T cell immune response against Encephalitozoon cuniculi infection

Gamma(delta) T cells have been reported to play an essential effector role during the early immune response against a wide variety of infectious agents. Recent studies have suggested that the gamma(delta) T cell subtype may also be important for the induction of adaptive immune response against certain microbial pathogens. In the present study, an early increase of gamma(delta) T cells during murine infection with Encephalitozoon cuniculi, an intracellular parasite, was observed. The role of gamma(delta) T cells against E. cuniculi infection was further evaluated by using gene-knockout mice. Mice lacking gamma(delta) T cells were susceptible to E. cuniculi infection at high challenge doses. The reduced resistance of delta(-/-) mice was attributed to a down-regulated CD8+ immune response. Compared with parental wild-type animals, suboptimal Ag-specific CD8+ T cell immunity against E. cuniculi infection was noted in delta(-/-) mice. The splenocytes from infected knockout mice exhibited a lower frequency of Ag-specific CD8+ T cells. Moreover, adoptive transfer of immune TCR(alpha)beta+ CD8+ cells from the delta(-/-) mice failed to protect naive CD8(-/-) mice against a lethal E. cuniculi challenge. Our studies suggest that gamma(delta) T cells, due to their ability to produce cytokines, are important for the optimal priming of CD8+ T cell immunity against E. cuniculi infection. This is the first evidence of a parasitic infection in which down-regulation of CD8+ T cell immune response in the absence of gamma(delta) T cells has been demonstrated.     

Counterion fluctuation and dielectric dispersion in linear polyelectrolytes

The thermal fluctuation in the concentration of counterions bound to a rodlike polyion was analyzed by expanding the fluctuation in a Fourier series along the rod. The amplitude and the relaxation time of fluctuations of various wave lengths were obtained as functions of the charge density and the length of the polyion. From these results the real and imaginary parts of the dielectric constant of the polyelectrolyte solution were derived as the sum of contributions of fluctuations of different modes. The dielectric dispersion curve or the Cole-Cole plot obtained was found to be in good agreement with experimental data.     

Estimation of bound quaternary ammoniumion to a rod-like polyion with a large alkyl residue

The amount of quaternary ammonium ion (Bu4N+), which is believed not to be bound to general carboxylpolyelectrolytes, bound to poly(iso BVE-co-MA) was estimated by conductivity measurements. In the region of the density of polyion charge in which the polyions are thought to take a free drain'ng conformation, it has been confirmed that the activity coefficient of Bu4N+ ions is less than 0.5 in the presence of a small amount of Bu4NCl, showing that the force between the counterions and the polyion is probably due to the hydrophobic interaction. Moreover, from the electrophoretic mobility Up of the polyion observed from the data of conductivity, it has been ascertained that Up of this polyion is two times larger than PAA, and the behavior of the quantity e/xiP with changing degree of ionization corresponds to that of the viscosity.     

DSC studies of the conformational stability of barstar wild-type.

The temperature induced unfolding of barstar wild-type of bacillus amyloliquefaciens (90 residues) has been characterized by differential scanning microcalorimetry. The process has been found to be reversible in the pH range from 6.4 to 8.3 in the absence of oxygen. It has been clearly shown by a ratio of delta HvH/delta Hcal near 1 that denaturation follows a two-state mechanism. For comparison, the C82A mutant was also studied. This mutant exhibits similar reversibility, but has a slightly lower transition temperature. The transition enthalpy of barstar wt (303 kJ mol-1) exceeds that of the C82A mutant (276 kJ mol-1) by approximately 10%. The heat capacity changes show a similar difference, delta Cp being 5.3 +/- 1 kJ mol-1 K-1 for the wild-type and 3.6 +/- 1 kJ mol-1 K-1 for the C82A mutant. The extrapolated stability parameters at 25 degrees C are delta G0 = 23.5 +/- 2 kJ mol-1 for barstar wt and delta G0 = 25.5 +/- 2 kJ mol-1 for the C82A mutant.     

Allowance for Spatial Dispersion of Dielectric Permittivity in Polyelectrolyte Model of DNA

Abstract

In order to allow for real dielectric properties of a solvent in calculating of electrostatic characteristics of strongly charged polyions such as DNA in salt solution we consider a simple model of linear dielectric response of a medium. The interactions between charged particles are treated in the framework of self-consistent-field approximation. The basic characteristic of the problem, electrostatic potential, can be found from the solution of non-linear integro-differential equation. Specifically we consider so-called quasimacroscopic model where dielectric response of a medium depends only on the distance from the polyion. Application of the approach for calculating of the B-to-Z free energy qualitatively retains the main conclusion obtained previously within the model with fixed dielectric constant: non-monotonous behavior of the free energy difference as a function of ionic strength. At the same time, essential sensitivity of the results to specific values of dielectric parameters is observed.     

Allowance for spatial dispersion of dielectric permittivity in polyelectrolyte model of DNA.

In order to allow for real dielectric properties of a solvent in calculating of electrostatic characteristics of strongly charged polyions such as DNA in salt solution we consider a simple model of linear dielectric response of a medium. The interactions between charged particles are treated in the framework of self-consistent-field approximation. The basic characteristic of the problem, electrostatic potential, can be found from the solution of non-linear integro-differential equation. Specifically we consider so-called quasimacroscopic model where dielectric response of a medium depends only on the distance from the polyion. Application of the approach for calculating of the B-to-Z free energy qualitatively retains the main conclusion obtained previously within the model with fixed dielectric constant: non-monotonous behavior of the free energy differences as a function of ionic strength. At the same time, essential sensitivity of the results to specific values of dielectric parameters is observed.     

Monte Carlo-Self Consistent Field Study of the Symmetrical Models of Polyelectrolytes

Abstract

Time saving procedures unifying Monte Carlo and self consistent field approaches for the calculation of equilibrium potentials and density distributions of mobile ions around a polyion in a polyelectrolyte system are considered. In the final version of the method the region around the polyion is divided into two zones—internal and external; all the ions of the internal zone are accounted for explicitly in a Monte Carlo procedure, in the external zone the self consistent field approximation is applied with an exchange of ions between regions. Simulations are carried out for cylindrical and spherical polyions in solutions with mono-and divalent ions and their mixtures. The results are compared with Poisson—Boltzmann approximation and experimental data on intrinsic viscosity.     

Gamma delta T cells are activated by polysaccharide K (PSK) and contribute to the anti-tumor effect of PSK

Polysaccharide K (PSK) is a widely used mushroom extract that has shown anti-tumor and immunomodulatory effects in both preclinical and clinical studies. Therefore, it is important to understand the mechanism of actions of PSK. We recently reported that PSK can activate toll-like receptor 2 and enhances the function of NK cells. The current study was undertaken to study the effect of PSK on gamma delta (γδ) T cells, another important arm of the innate immunity. In vitro experiments using mouse splenocytes showed that γδ T cells produce IFN-γ after treatment with PSK and have up-regulated expression of CD25, CD69, and CD107a. To investigate whether the effect of PSK on γδ T cells is direct or indirect, purified γδ T cells were cultured either alone or together with bone marrow-derived DC in a co-culture or trans-well system and then stimulated with PSK. Results showed that direct cell-to-cell contact between γδ T cells and DC is required for optimal activation of γδ T cells. There was also reciprocal activation of DC by PSK-activated γδ T cells, as demonstrated by higher expression of costimulatory molecules and enhanced production of IL-12 by DC in the presence of γδ T cells. PSK can also co-stimulate γδ T cells with anti-TCR and anti-CD3 stimulation, in the absence of DC. Finally, in vivo treatment with PSK activates γδ T cells among the tumor infiltrating lymphocytes, and depleting γδ T cells during PSK treatment attenuated the anti-tumor effect of PSK. All together, these results demonstrated that γδ T cells are activated by PSK and contribute to the anti-tumor effect of PSK.     

A novel alpha-proton exchange reaction catalyzed by Escherichia coli methionyl-tRNA synthetase.

The Escherichia coli truncated methionyl-tRNA synthetase (delta MTS) was shown to catalyze alpha-carbon hydrogen-deuterium exchange of L-selenomethionine, L-methionine, L-ethionine, and L-norleucine in the presence of deuterium oxide. The rate of alpha-proton exchange for L-methionine was shown to be linear with respect to delta MTS concentration. The exchange reaction showed saturation kinetics with apparent Km values of 21 and 4 mM in the absence and presence of saturating adenosine concentrations, respectively. As expected, delta MTS did not catalyze alpha-proton exchange of D-methionine since the enzyme has been shown to be specific for L-amino acids. In the absence of enzyme or in the presence of an equivalent concentration of Zn2+, no hydrogen-deuterium exchange was detected. The exchange reaction was not observed with L-methioninol, an analogue of L-methionine lacking the carboxylate group. These results suggest that the alpha-carboxylate group is a requirement for the delta MTS-catalyzed exchange reaction. The E. coli methionyl-tRNA synthetase (MTS) has previously been shown to be a zinc metalloprotein [Posorske, L. H., Cohn, M., Yanagisawa, N., & Auld, D. S. (1979) Biochim. Biophys. Acta 576, 128]. On the basis of the structural and mechanistic information available on MTS, we propose that the enzyme-bound zinc coordinates the carboxylate of the amino acid, while a base on the enzyme is responsible for exchange of the alpha-proton. The role of the enzyme-bound metal is to render the alpha-proton more acidic through coordination of the carboxylate group.(ABSTRACT TRUNCATED AT 250 WORDS)