Observation of enhanced transmission for s-polarized light through a subwavelength slit.

Enhanced optical transmission (EOT) through a single aperture is usually achieved by exciting surface plasmon polaritons with periodic grooves. Surface plasmon polaritons are only excited by p-polarized incident light, i.e. with the electric field perpendicular to the direction of the grooves. The present study experimentally investigates EOT for s-polarized light. A subwavelength slit surrounded on each side by periodic grooves has been fabricated in a gold film and covered by a thin dielectric layer. The excitation of s-polarized dielectric waveguide modes inside the dielectric film strongly increases the s-polarized transmission. A 25 fold increase is measured as compared to the case without the dielectric film. Transmission measurements are compared with a coupled mode method and show good qualitative agreement. Adding a waveguide can improve light transmission through subwavelength apertures, as both s and p-polarization can be efficiently transmitted.

Electrostatic properties of two porin channels from Escherichia coli.

The electrostatic interactions in the channels of OmpF and PhoE porins from Escherichia coli were analysed on the basis of a macroscopic multi-dielectric model of the protein-membrane complex derived from the respective porin X-ray structures. The membrane was represented as layers of distinct dielectric constants corresponding to the aliphatic core and the polar head groups of the lipids. The pKa values of the titratable groups and the electrostatic field in the region of the channel were calculated by the finite difference technique. In spite of the differences in sequences and charge constellations, the calculated electrostatic properties of the two porins are similar in several aspects: (1) unusual titration behaviour (pKa below 7) was found for some groups of the cluster of basic residues at the constriction of the pore; (2) a number of acidic groups buried between the internal loop and the barrel wall are stabilized in their protonated forms at neutral pH; (3) there is a strong transverse electrostatic field in the channel characterized by a screw-like form. The strength of the field is greatest at the region of the constriction zone. This would facilitate the diffusion of solutes with a large dipole moment such as free amino acids. Differences between the electrostatic fields of OmpF and PhoE are mainly confined to that end of the pore that faces the cell exterior in vivo. In OmpF the electrostatic potential is close to zero in this region of the channel, whereas a positive potential was found in PhoE. It was shown that the experimentally observed difference in ion selectivity of the two porins can largely be attributed to this distinct electrostatic property.

pK(a) Calculations suggest storage of an excess proton in a hydrogen-bonded water network in bacteriorhodopsin.

Calculations of protonation states and pK(a) values for the ionizable groups in the resting state of bacteriorhodopsin have been carried out using the recently available 1.55 A resolution X-ray crystallographic structure. The calculations are in reasonable agreement with the available experimental data for groups on or near the ion transport chain (the retinal Schiff base; Asp85, 96, 115, 212, and Arg82). In contrast to earlier studies using lower-resolution structural data, this agreement is achieved without manipulations of the crystallographically determined heavy-atom positions or ad hoc adjustments of the intrinsic pK(a) of the Schiff base. Thus, the theoretical methods used provide increased reliability as the input structural data are improved. Only minor effects on the agreement with experiment are found with respect to methodological variations, such as single versus multi-conformational treatment of hydrogen atom placements, or retaining the crystallographically determined internal water molecules versus treating them as high-dielectric cavities. The long-standing question of the identity of the group that releases a proton to the extracellular side of the membrane during the L-to-M transition of the photocycle is addressed by including as pH-titratable sites not only Glu204 and Glu194, residues near the extracellular side that have been proposed as the release group, but also an H(5)O(2)(+) molecule in a nearby cavity. The latter represents the recently proposed storage of the release proton in an hydrogen-bonded water network. In all calculations where this possibility is included, the proton is stored in the H(5)O(2)(+) rather than on either of the glutamic acids, thus establishing the plausibility on theoretical grounds of the storage of the release proton in bacteriorhodopsin in a hydrogen-bonded water network. The methods used here may also be applicable to other proteins that may store a proton in this way, such as the photosynthetic reaction center and cytochrome c oxidase.

Electrostatic coupling to pH-titrating sites as a source of cooperativity in protein-ligand binding.

This paper describes an alternative mechanism for the cooperative binding of charged ligands to proteins. The ligand-binding sites are electrostatically coupled to protein side chains that can undergo protonation and deprotonation. The binding of one ligand alters the protein's protonation equilibrium in a manner that makes the the binding of the second ligand more favorable. This mechanism requires no conformational change to produce a cooperative effect, although it is not exclusive of conformational change. We present a theoretical description of the mechanism, and calculations on three kinds of systems: A model system containing one protonation site and two ligand-binding sites; a model system containing two protonation sites and two ligand-binding sites; and calbindin D9k, which contains two Ca2+-binding sites and 30 protonation sites. For the one-protonation-site model, it is shown that the influence of the protonation site can only be cooperative. The competition of this effect with the anticooperative effect of ligand-ligand repulsion is studied in detail. For the two-protonation site model, the effect can be either cooperative or, in special cases, anticooperative. For calbindin D9k, the calculations predict that six protonation sites in or near the ligand-binding sites make a cooperative contribution that approximately cancels the anticooperative effect of Ca2+-Ca2+ repulsion, accounting for more than half of the total cooperative effect that is needed to overcome repulsion and produce the net cooperativity observed experimentally. We argue that cooperative mechanisms of the kind described here are likely when there is more than one ligand-binding site in a protein domain.

Optimization of the electrostatic interactions in proteins of different functional and folding type.

The 3-dimensional optimization of the electrostatic interactions between the charged amino acid residues was studied by Monte Carlo simulations on an extended representative set of 141 protein structures with known atomic coordinates. The proteins were classified by different functional and structural criteria, and the optimization of the electrostatic interactions was analyzed. The optimization parameters were obtained by comparison of the contribution of charge-charge interactions to the free energy of the native protein structures and for a large number of randomly distributed charge constellations obtained by the Monte Carlo technique. On the basis of the results obtained, one can conclude that the charge-charge interactions are better optimized in the enzymes than in the proteins without enzymatic functions. Proteins that belong to the mixed alpha beta folding type are electrostatically better optimized than pure alpha-helical or beta-strand structures. Proteins that are stabilized by disulfide bonds show a lower degree of electrostatic optimization. The electrostatic interactions in a native protein are effectively optimized by rejection of the conformers that lead to repulsive charge-charge interactions. Particularly, the rejection of the repulsive contacts seems to be a major goal in the protein folding process. The dependence of the optimization parameters on the choice of the potential function was tested. The majority of the potential functions gave practically identical results.

The optimization of protein-solvent interactions: thermostability and the role of hydrophobic and electrostatic interactions.

Protein-solvent interactions were analyzed using an optimization parameter based on the ratio of the solvent-accessible area in the native and the unfolded protein structure. The calculations were performed for a set of 183 nonhomologous proteins with known three-dimensional structure available in the Protein Data Bank. The dependence of the total solvent-accessible surface area on the protein molecular mass was analyzed. It was shown that there is no difference between the monomeric and oligomeric proteins with respect to the solvent-accessible area. The results also suggested that for proteins with molecular mass above some critical mass, which is about 28 kDa, a formation of domain structure or subunit aggregation into oligomers is preferred rather than a further enlargement of a single domain structure. An analysis of the optimization of both protein-solvent and charge-charge interactions was performed for 14 proteins from thermophilic organisms. The comparison of the optimization parameters calculated for proteins from thermophiles and mesophiles showed that the former are generally characterized by a high degree of optimization of the hydrophobic interactions or, in cases where the optimization of the hydrophobic interactions is not sufficiently high, by highly optimized charge-charge interactions.

Optimization of the electrostatic interactions between ionized groups and peptide dipoles in proteins.

The three-dimensional optimization of the electrostatic interactions between the charged amino acid residues and the peptide partial charges was studied by Monte-Carlo simulations on a set of 127 nonhomologous protein structures with known atomic coordinates. It was shown that this type of interaction is very well optimized for all proteins in the data set, which suggests that they are a valuable driving force, at least for the native side-chain conformations. Similar to the optimization of the charge-charge interactions (Spassov VZ, Karshikoff AD, Ladenstein R, 1995, Protein Sci 4:1516-1527), the optimization effect was found more pronounced for enzymes than for proteins without enzymatic function. The asymmetry in the interactions of acidic and basic groups with the peptide dipoles was analyzed and a hypothesis was proposed that the properties of peptide dipoles are a factor contributing to the natural selection of the basic amino acids in the chemical composition of proteins.

Effects of GABAB receptor antagonists on learning and memory retention in a rat model of absence epilepsy.

A variety of animal models of absence epilepsy have been described and among these exists a genetically susceptible strain of rat (genetic absence epilepsy rats of Strasbourg (GAERS)). These rats produce periods of behavioural arrest with simultaneous production of cortical spike and wave discharges (SWD). GABAB receptor antagonists suppress completely the production of these spike and wave discharges. GABAB receptor ligands have also been reported to affect cognitive performance in rodents. The present study examined the cognitive performance of GAERS and the influence of GABAB receptor antagonists on this activity. Rats were injected intraperitoneally once per day with saline or a GABAB receptor antagonist (CGP 36742 (3-amino-propyl-n-butyl-phosphinic acid) 100 mg/kg; CGP 56433 ([3-(1-(S)-[(3-(cyclohexylmethyl)hydroxy phosphinyl]-2-(S) hydroxy propyl] amino]ethyl]benzoic acid) ([3-{1-(S)-[{3-(cyclohexylmethyl)hydroxy phosphinyl]-2-(S) hydroxy propyl] amino]ethyl]benzoic acid) 1 mg/kg or CGP 61334 ([3-({[3-[(diethoxymethyl)hydroxy phosphinyl]propyl] amino}methyl]-benzoic acid (1 mg/kg). A two-way active avoidance test paradigm with negative reinforcement was used. Untreated GAERS performed significantly better than non-epileptic rats (P < 0.05) and this enhancement in cognitive performance was sustained in rats treated with the GABAB receptor antagonists.

Effects of GABA(B) receptor antagonists on spontaneous and on GABA-induced mechanical activity of guinea-pig smooth muscle preparations.

The majority of GABA(B) receptor antagonists have been based on alterations of the acidic moiety of gamma-aminobutyric acid (GABA) or baclofen, such as the first selective antagonist phaclofen. More recently, a new structural class of compounds derived by p-alkyl substitution in the phosphinic analog of GABA, such as CGP35348 (3-amino-propyl-(diethoxymethyl)-phosphinic acid), have been introduced as GABA(B) receptor antagonists. The present study examine the influence of a series of structurally related phosphinic acid analogues on mechanical activity and their effect on GABA-induced reactions in ileal smooth muscle. In our experiments, GABA exerted a biphasic contractile-relaxation effect with pronounced dose-dependent characteristics. 3-[[1-(S)-(3,4-Dihydrophenyl) ethyl]amino]-2-(S)-hydroxy-propyl]-(phenylmethyl)-phosphinic acid hydrochloride (CGP55845A) induced prolonged relaxation without changing the phasic activity of the ileum preparations. [3-[1-R-[[2-(S)-hydroxy-3-[hydroxy-4-methoxyphenyl]-methyl]-phosphinyl]-propyl]-aminoethyl]-benzoic acid (CGP62349) did not change the mechanical activity of smooth muscle preparation. Trans 3-[6-[[Cyclo hexylmethyl-hydroxy-phosphinyl]-methyl]-3-morpholinyl]-benzoic acid (CGP71982) itself induced smooth muscle contractions. GABA(B) receptor antagonists decreased concentration-dependently the relaxation phase of the action of GABA from 50% to 90%. Their effect on the contractile phase of the action of GABA was quite different-CGP55845A decreased it dose-dependently, whereas CGP62349 and CGP71982 did not change it significantly. These findings prompted us to assume that the GABA(B) receptor antagonists studied, being phosphinic analogues, probably act on GABA(B) receptors in guinea-pig ileum smooth muscles.

Urea unfolding and stability of gamma-II crystallin.

The conformational stability of gamma-II crystallin at pH 7.0 was estimated by studying its urea denaturation at isothermal conditions. The conformational states were monitored by far UV-CD and fluorescence measurements. Gamma-II crystallin shows sigmoidal order-disorder transition curves by both methods. The presence of more than one intermediate was confirmed but at neutral pH. The experiment results were critically analyzed in terms of both linear extrapolation and Tanford's models. The Gibbs free energy of unfolding delta G u,H2O = -36 kcal mol-1 was obtained. This value corresponds to the high conformational stability of the protein predicted qualitatively by its crystal structure.

Computer simulation of excitation-contraction coupling in cardiac muscle. A study of the regulatory role of calcium binding to troponin C.

The influence of a change of troponin concentration as well of a change of binding and dissociation of Ca2+ ions to the regulatory protein troponin C on the time course of isometric tension has been studied, using a mathematical model developed to investigate excitation-contraction coupling in cardiac muscle cells. The numerical simulations show that peak amplitude, rate of force development, time to peak tension and relaxation time depend significantly on the above parameters even in the case when the equilibrium dissociation constant remains unchanged. The obtained results might be useful for the planing of new experiments in the view of the fact that no similar data have been reported for cardiac muscle cells as yet.

Influence of tacrine on dopamine-induced reactions of the gastric smooth muscle of rats.

The clinical usage of the cholinesterase inhibitor tacrine for treatment of Alzheimer's disease is accompanied by adverse effects on the gastrointestinal tract. These adverse effects are a result of the direct action of tacrine on the intestinal smooth muscles or of the modulation of certain neurotransmitters regulating gastrointestinal functions. Dopamine is a neurotransmitter that modulates gastrointestinal motility. This study was designed to examine in vitro the effects of tacrine on dopamine-induced changes in spontaneous activity of smooth muscle preparations from rat's gastric corpus. The mechanical activity was isometrically registered. Tacrine 1.10(-7)-1.10(-5) mol/l caused smooth muscle contraction, which was blocked by atropine 1.10(-6) mol/l. Tacrine 1.10(-4) mol/l provoked a relaxation resistant to atropine. Dopamine and D(2)-receptor antagonists haloperidol and R121 had no effect on tacrine-induced relaxation. Dopamine-induced contraction was concentration-dependent. It was blocked by D(2)-receptor antagonists haloperidol and R121 and by tacrine 1.10(-4) mol/l. In the presence of tacrine 1.10(-7)-10(-5) mol/l or atropine the dopamine-induced contraction was significant. The data obtained suggested that tacrine 1.10(-4) mol/l inhibited the dopamine effects on gastric corpus smooth muscles. The effect was probably not dependent on its anticholinesterase activity or not realized through direct influence on D(2)-dopamine receptors.

Affinity of a 1,4-benzodioxane derivative with alpha-adrenolytic activity to isolated synaptic membranes from rat brain.

The experiments carried out with KS 5-2, dibozan and WB 4101 allowed for a comparison of the affinity of KS 5-2 with that of the other two compounds. High affinity of the three compounds and differences in their affinity with respect to the alpha-adrenergic receptors were established. The results were compared with the 1H-NMR spectroscopic data, revealing the intimate structure of WB 4101, dibozan and KS 5-2 in such a way as to substantiate their pharmacological action by their chemical structure.

Spatial optimization of electrostatic interactions between the ionized groups in globular proteins.

A model approach is suggested to estimate the degree of spatial optimization of the electrostatic interactions in protein molecules. The method is tested on a set of 44 globular proteins, representative of the available crystallographic data. The theoretical model is based on macroscopic computation of the contribution of charge-charge interactions to the electrostatic term of the free energy for the native proteins and for a big number of virtual structures with randomly distributed on protein surface charge constellations (generated by a Monte-Carlo technique). The statistical probability of occurrence of random structures with electrostatic energies lower than the energy of the native protein is suggested as a criterion for spatial optimization of the electrostatic interactions. The results support the hypothesis that the folding process optimizes the stabilizing effect of electrostatic interactions, but to very different degree for different proteins. A parallel analysis of ion pairs shows that the optimization of the electrostatic term in globular proteins has increasingly gone in the direction of rejecting the repulsive short contacts between charges of equal sign than of creating of more salt bridges (in comparison with the statistically expected number of short-range ion pairs in the simulated random structures). It is observed that the decrease in the spatial optimization of the electrostatic interactions is usually compensated for by an appearance of disulfide bridges in the covalent structure of the examined proteins.

Relationship between accessibility and reactivity of Lys, Met and Tyr in subtilisins DY and Carlsberg.

The chemical reactivities of lysyl, tyrosyl and methionyl residues in subtilisin DY are compared with the computed accessibilities to the water molecules of the corresponding sensitive atoms in the crystal structure of subtilisin Carlsberg. A good correlation between accessibilities and reactivity was established for the nonionized specific reagents glyceraldehyde and chloramine T used for modification of lysine and methionine residues, respectively. In a few cases an apparent deviation from the general relationship was established. The disagreement may indicate that differences between crystal and solution structures exist with altered accessibilities of the atoms under consideration. However, when an electrically charged system takes part in the reaction and it is in low concentration, as in the case of tetranitromethane treatment where the phenoxylate anion reacts, the correlation is more complex. In this case not only the accessibility, but also the electrostatic field around the reacting group, should be taken into account for the explanation of the reaction observed.

Theoretical model and computer simulation of excitation-contraction coupling of mammalian cardiac muscle.

A mathematical model is developed to investigate the kinetics of electrical, mechanical and molecular processes in mammalian cardiac muscle. Isometric contractions at different muscle length and frequency of stimulation in response to a rhythmically applied clamp pulse or artificial action potential are simulated. Numerical results show that concentration of Ca2+ ions, bound to Ca(2+)-specific sites on protein troponin C, could be a regulatory factor in actin-myosin interactions and subsequent production of force in Huxley's mathematical approach for the sliding mechanism. The behavior of the model is compared to that of living cardiac muscle.

Effect of insulin and glucagon on the mobility of ESR-probes incorporated in rat liver plasma membranes.

1. The effect of different concentrations of insulin (INS) and glucagon (GLU) on the rotational mobility of a membrane-incorporated spin probe 2,2-6,6-tetramethyl-4-capriloyl piperidine-1-oxil (C7) was investigated by electron spin resonance (ESR) technique. 2. Two strong adenylate cyclase effectors guanosine 5'-triphosphate (GTP) and guanylyl 5'-imidodiphosphate [Gpp(NH)p], as well as an antioxidant 4-methyl-2,6-ditretbutilphenol (AO) and a nonprotein hormone prostaglandin E2 (PGE2) were used as reference effectors. 3. Applied effectors reduced by 30-82% the rotation correlation time (TR) of the rat liver plasma membranes spin probe C7. The effect was time-dependent and reached saturation 30-40 min after the effector application. 4. The kinetic- and concentration-dependent changes in TR were described by a simple phenomenological model. The apparent binding constants and the number of the apparent membrane binding sites of the effectors used were calculated using the model.

Structural modeling and electrostatic properties of aspartate transcarbamylase from Saccharomyces cerevisiae.

In Saccharomyces cerevisiae the first two reactions of the pyrimidine pathway are catalyzed by a multifunctional protein which possesses carbamylphosphate synthetase and aspartate transcarbamylase activities. Genetic and proteolysis studies suggested that the ATCase activity is carried out by an independently folded domain. In order to provide structural information for ongoing mutagenesis studies, a model of the three-dimensional structure of this domain was generated on the basis of the known X-ray structure of the related catalytic subunit from E. coli ATCase. First, a model of the catalytic monomer was built and refined by energy minimization. In this structure, the conserved residues between the two proteins were found to constitute the hydrophobic core whereas almost all the mutated residues are located at the surface. Then, a trimeric structure was generated in order to build the active site as it lies at the interface between adjacent chains in the E. coli catalytic trimer. After docking a bisubstrate analog into the active site, the whole structure was energy minimized to regularize the interactions at the contact areas between subunits. The resulting model is very similar to that obtained for the E. coli catalytic trimer by X-ray crystallography, with a remarkable conservation of the structure of the active site and its vicinity. Most of the interdomain and intersubunit interactions that are essential for the stability of the E. coli catalytic trimer are maintained in the yeast enzyme even though there is only 42% identity between the two sequences. Free energy calculations indicate that the trimeric assembly is more stable than the monomeric form. Moreover an insertion of four amino acids is localized in a loop which, in E. coli ATCase, is at the surface of the protein. This insertion exposes hydrophobic residues to the solvent. Interestingly, such an insertion is present in all the eukaryotic ATCase genes sequences so far, suggesting that this region is interacting with another domain of the multifunctional protein.

A seven-years experience in treatment of childhood ALL in Bulgaria.

After analyzing nonsatisfactory therapeutic results in the 1970s and early 1980s, the 81-01 treatment protocol of the Dana-Farber Cancer Institute was initiated in 1987 in the Children's Oncohaematology Clinic in Sofia, Bulgaria. Two hundred thirty patients were enrolled with a period of observation of a minimum of 14 and a maximum of 97 months; the median age was 5.83 +/- 3.6 years. According to the original criteria, standard risk (SR) patients were 38.26% and high-risk (HR) patients 61.74%. The probability for event-free survival at the seventh year estimated with the Kaplan-Meier method for the total group was 0.67 +/- 0.04 (+/- SE) and 0.55 +/- 0.09 and 0.81 +/- 0.06 for HR and SR, respectively (P < .001). Improvement of therapeutic results in terms of remission failures, early deaths, patients lost to follow-up, and rate of relapses is discussed.