Point Mutations of Nicotinic Receptor α1 Subunit Reveal New Molecular Features of G153S Slow-Channel Myasthenia

Slow-channel congenital myasthenic syndromes (SCCMSs) are rare genetic diseases caused by mutations in muscle nicotinic acetylcholine receptor (nAChR) subunits. Most of the known SCCMS-associated mutations localize at the transmembrane region near the ion pore. Only two SCCMS point mutations are at the extracellular domains near the acetylcholine binding site, α1(G153S) being one of them. In this work, a combination of molecular dynamics, targeted mutagenesis, fluorescent Ca²⁺ imaging and patch-clamp electrophysiology has been applied to G153S mutant muscle nAChR to investigate the role of hydrogen bonds formed by Ser 153 with C-loop residues near the acetylcholine-binding site. Introduction of L199T mutation to the C-loop in the vicinity of Ser 153 changed hydrogen bonds distribution, decreased acetylcholine potency (EC₅₀ 2607 vs. 146 nM) of the double mutant and decay kinetics of acetylcholine-evoked cytoplasmic Ca²⁺ rise (τ 14.2 ± 0.3 vs. 34.0 ± 0.4 s). These results shed light on molecular mechanisms of nAChR activation-desensitization and on the involvement of such mechanisms in channelopathy genesis.     

The exterior magnetic field for the multilayer ellipsoidal model of the brain

The magnetic induction field in the exterior of an ellipsoidallyinhomogeneous, four-conducting-layer model of the human headis obtained analytically up to its quadrupole approximation.The interior ellipsoidal core represents the homogeneous brainwhile each one of the shells represents the cerebrospinal fluid,the skull and the scalp, all characterized by different conductivities.The inhomogeneities of these four domains, together with theanisotropy imposed by the use of the ellipsoidal geometry, providethe most realistic physical and geometrical model of the brainfor which an analytic solution of the biomagnetic forward problemis possible. It is shown that in contrast to the spherical model,where shells of different conductivity are magnetically invisible,the magnetic induction field in ellipsoidal geometry is stronglydependent on the conductivity supports. The fact that sphericalshells of different conductivity are invisible has enhancedthe common belief that the biomagnetic forward solution doesnot depend on the conductivity profiles. As we demonstrate inthe present work, this is not true. Hence, the proposed multilayeredellipsoidal model provides a qualitative improvement of therealistic interpretation of magnetoencephalography (MEG) measurements.We show that the presence of the shells of different conductivitycan be incorporated in the form of the dipole vector for thehomogeneous model. Numerical investigations show that the effectsof shell inhomogeneities are almost as sound as the level ofMEG measurements themselves. The degenerate cases, where eitherthe differences of the conductivities within the shells disappear,or the ellipsoidal geometry is reduced to the spherical one,are also considered.     

Structural Analogy Method in Studies of Adamantanes

The potential of the structural analogy method in the topological studies of adamantane derivatives and analogs is demonstrated. The topological and structural characteristics of these compounds are calculated. Chromatographic retention indices were determined experimentally. Correlations between the topological indices and the physicochemical and chromatographic characteristics are investigated.     

Photoacclimation in spathiphyllum

We studied photoacclimation in Spathiphyllum grown at an irradiance of 40 or 420 micromol/m2 s (LL or HL, respectively). All parameters studied responded to acclimation. Leaves at LL, in contrast to HL, were thinner and oriented perpendicular to the incident light, had more chlorophyll per g f w, fewer stomata on the upper leaf surface and a reduced layer of mesophyll cells. Their chloroplasts at HL had wider grana with less thylakoids per granum, and better organized photosystems than at LL. PSI and PSII activities per mg chlorophyll ( Vmax ), and PSI and PSII content (total activity per g f w), were lower at LL than at HL and so was the light requirement for saturation of the PSI or PSII partial photoreactions, suggesting that fewer photosystems with larger antenna size prevail at LL, but many more with smaller antenna size at HL. Analysis of chlorophyll distribution among the thylakoid pigment-protein complexes showed less antenna chlorophyll serving PSII (CPa+LHCP1+LHCP3) than that serving PSI (CPIa+CPI+LHCP2) at LL as compared to HL, and thus a lower PSII/PSI ratio at LL, in agreement with the general finding that LL plants, with larger PSII antenna size, have lower PSII/PSI ratio. The increase in PSI antenna size at LL was correlated with the increase in the distribution of chlorophyll in pigment-protein complexes serving PSI, and a very large chlorophyll/protein molar ratio in the isolated CPI complex. On the other hand, the PSII antenna chlorophyll (CPa+LHCP1+LHCP3) on a g f w basis, and the chlorophyll a/b ratio remained more or less constant at LL or HL. This may reflect our finding that Spathiphyllum contains mainly the 27 kDa inner LHCII antenna protein, the size of which remains unaffected by photoacclimation. The increase in the distribution of chlorophyll in pigment-protein complexes serving PSII at HL, therefore, reflects the higher population of PSII at HL. Very high PSI activity was found at HL, which we attribute to the highly organized small in size PSI.     

Direct zonal liquid chromatographic method for the kinetic study of actinomycin-DNA binding

The binding of an anticancer drug (actinomycin D or ACTD) to double-stranded DNA (dsDNA) was studied by means of high-performance liquid chromatography (HPLC). ACTD is an antitumor antibiotic containing one chromophore group and two pentapeptidic lactone cycles that binds dsDNA. Incubations of ACTD with DNA were performed at physiological pH. The complexed and free ligand concentrations of the mixture were quantified at 440 nm from their separation on a size-exclusion chromatographic (SEC) column using the same buffer for the elution and the sample incubation. The DNA and the ACTD-DNA complexes were eluted at the column exclusion volume while the ligand was retained on the support. An apparent binding curve was obtained by plotting the amount emerging at the exclusion column volume against that eluted at free ACTD retention volume. A dissociating effect was evidenced and the binding parameters were significantly different from those obtained at equilibrium by visible absorbance titration. The equilibrium binding parameters determined by absorption spectroscopy were used as starting data in the numerical simulations of the chromatographic process. The results showed a strong dependency of the apparent binding parameters on the reaction kinetics. Finally the comparison of the apparent binding curve obtained from the HPLC experiments and from the numerical simulations permitted an evaluation of the dissociation rate constant (kd = 0.004 s(-1)).     

Gas chromatography of halogenated adamantanes

The main thermodynamic characteristics of the adsorption of haloadamantanes on graphitized thermal carbon black were determined by experimental measurements and by calculations in terms of molecular-statistic theory of adsorption. Using experimental data, the Kovac retention indices were calculated and the optimum conditions for gas-chromatographic separation of haloadamantanes on stationary phases with different polarity were elucidated. The influence of the cage effect in the adamantane unit on the chromatographic properties of haloadamantanes was established.     

Calculating a function of a matrix with a real spectrum

Let T be a square matrix with a real spectrum, and let f be an analytic function. The problem of the approximate calculation of f(T) is discussed. Applying the Schur triangular decomposition and the reordering, one can assume that T is triangular and its diagonal entries t_ii are arranged in increasing order. To avoid calculations using the differences t_ii ? t_jj with close (including equal) t_ii and t_jj, it is proposed to represent T in a block form and calculate the two main block diagonals using interpolating polynomials. The rest of the f(T) entries can be calculated using the Parlett recurrence algorithm. It is also proposed to perform some scalar operations (such as the building of interpolating polynomials) with an enlarged number of significant decimal digits.

     

Extraction recovery of vanadium(V) from sulfuric acid solutions

The IR and electronic absorption spectra of di-2-ethylhexyl hydrogen phosphate (HDEHP) extracts of vanadium(V) and sulfuric acid and of vanadium(V) solutions in sulfuric acid were studied. The composition of the extractable complex was determined, and the equation of vanadium(V) extraction with HDEHP was suggested. The equilibrium constant of vanadium(V) extraction from concentrated sulfuric acid solutions was found.