Effect of Phosphatidyl Serine Decarboxylase on Neural Excitation

IT has been proposed that phosphatidyl serine (PS) plays a central role in neural excitation^1–4. Consequently, there have been numerous reports of the interaction of isolated PS with ions, local anaesthetics and anionic and cationic nerve blocking agents^5–11. These results parallel those obtained with isolated nerve axons^12–14. There have been conflicting reports as to whether the carboxyl or phosphate portions of the PS molecule provide the biologically important cation exchange sites^5,6,9. In the case of the squid axon, ion substitution experiments indicate that these sites on the external membrane surface are carboxyl in nature¹⁵. To obtain direct evidence for the involvement of the carboxyl groups of PS in neural excitation, we determined the effect of PS decarboxylase on neural activity.     

Ionic transporting systems of skeletal muscle in relation with innervation and their involvement in myotonic diseases

Excitation-contraction coupling describes the series of events that begins with propagated action potential on the muscle fiber surface membrane and leads to the twitch contraction of the fiber. The generation of an action potential during excitation requires rapid sequential changes in membrane conductances of Na⁺, Ca²⁺, and K⁺ ions that depend upon the opening and closing of the respective channels. Myotonic disorders are inherited diseases whose clinical manifestations include electrophysiological signs such as increased excitability and delayed relaxation of the muscles after voluntary contraction. All these disorders appears to be due to an abnormality of the muscle itself since they persist after section or blocking of the motor nerve after curarization. Most experimental and clinical data suggest that human myotonia arises from genetically-induced structural and functional alterations of the muscle membrane. The purpose of this article is to focus on the more recent developments in the molecular and pharmacological analysis of cation transporting systems such as ionic channels and (Na⁺, K⁺) ATPase in myotonic disorders.Special issue dedicated to Dr. Lawrence Austin.     

Micellar‐mediated binding interaction between proflavine hemisulfate and salicylic acid: Spectroscopic insights and its analytical application

The molecular interactions between salicylic acid (SA) and proflavin hemisulfate (PF) were investigated using fluorescence and UV–VIS absorption spectroscopy in an aqueous micellar environment. Changes in the absorption spectra of SA in the presence of PF indicate a ground state interaction between salicylate and proflavine hemisulfate ions to form a complex. The excitation bands of SA monitored at its emission wavelength reveal a red spectral shift of 8390.54 and 2037.75 cm^‐1 when compared with absorption bands. The intensity of both excitation bands decreased in the presence of increasing amounts of PF. The absence of excitation bands of PF rules out the possibility of its direct excitation and suggests energy transfer from excited SA to PF, resulting in quenching of the SA fluorescence. The fluorescence quenching results were found to fit the well‐known Stern–Volmer (S–V) relation. S–V plots at different temperatures were used to further evaluate thermodynamic parameters such as ?G, ?H and ΔS. The thermodynamic and kinetic data obtained from the quenching results were used to investigate the possible mechanism of binding, the nature of the binding force and the distance between SA and PF molecules. The linear relation between SA fluorescence quenching and PF concentration used to develop an analytical method for the determination of PF from Lorexane (a veterinary cream) using a fluorescence quenching method. Copyright © 2012 John Wiley & Sons, Ltd.     

The kinetics of carotenoid absorption changes in intact cells of photosynthetic bacteria

The kinetics of carotenoid absorption changes have been measured in intact cells of Rhodopseudomonas capsulata after short flash excitation. The observed changes were consistent with the thesis that they indicate the development and dissipation of membrane potential. In the generation of the absorption changes in anaerobic cells, fast (complete in 0.5 ms) and slow (half-time 3 ms) components can be distinguished. The slow component corresponds kinetically to the rate of cytochrome c re-reduction and is similarly antimycin sensitive. These data are similar to those observed in isolated chromatophores which have been artifically poised with redox mediators. In aerobic intact cells the kinetic profile is altered, mainly because the decay of the carotenoid change is much faster. Inhibition of respiration with KCN leads to flash-induced changes similar to those in anaerobic cells. At least two components can be distinguished in the decay of the carotenoid absorption changes in anaerobic intact cells. Only the faster decay component was inhibited by venturicidin which suggests that it corresponds to H⁺ flux through the F₀F₁-ATPase during ATP synthesis. The contribution of the venturicidin-sensitive decay to the total decay was dependent upon the initial amplitude of the carotenoid absorption change produced by the flash group. This suggests that there is an apparent threshold of membrane potential for ATP synthesis. Supporting evidence was provided by the finding that venturicidin stimulated the steady-state light-induced carotenoid absorption change at high but not at low light intensities. The entire decay of the carotenoid absorption changes was stimulated by carbonyl cyanide p-trifluoromethoxyphenylhydrazone in a manner that can be interpreted as an ionophore catalysing the dissipation of membrane potential.     

Electric-Field-Induced Shifts in the Infrared Spectrum of Conducting Nerve Axons

Difference spectra between resting and excited nerve in the infrared region between 2000 and 1000 cm^-1 have been examined with a resolution of 0.5 cm^-1. Spectra were obtained with a modified Perkin-Elmer model 521 grating infrared spectrophotometer (Perkin-Elmer Corp., Instrument Div., Norwalk, Conn.), and the signal-to-noise (S/N) ratio was improved by time averaging and digital smoothing. Peaks occurring in the regions around 1030 and 1066 cm^-1 are identified as P-O-C stretch, at 1410-1414 cm^-1 as C-H deformation, and at 1750 cm^-1 as carbonyl stretch. The difference peaks appear to be due to a shift of about 1 cm^-1 in the absorption band to lower frequencies for the three lower frequency bands and to a higher frequency for 1750 cm^-1 band. Since the difference peaks appear when the nerve is modulated by a propagated action potential it is concluded that the changing electrical field across the nerve membrane is perturbing the absorption spectrum. From evidence presented it appears likely that these difference peaks are due to phospholipids in the nerve membrane and that they may be related to conformational changes associated with membrane permeability.     

Mechanistic and conformational studies on the interaction of sulfamethazine with human immunoglobulin G by molecular modeling and multi‐spectroscopic approach in vitro

Binding interaction of sulfamethazine (SMZ) with human immunoglobulin G (HIgG) has been explored under physiological conditions. The interaction mechanism was firstly predicted through molecular modeling which showed that several hydrogen bonds participated in stabilizing the SMZ ? HIgG complex. Fluorescence spectroscopy, ultraviolet–visible (UV–vis) light absorption and circular dichroism (CD) spectroscopy were used to analyze the binding site, binding constants and effects of SMZ on HIgG stability and secondary structure. The binding parameters and thermodynamic parameters at different temperatures for the reaction have been calculated according to the Scatchard, Sips and Van 't Hoff equations, respectively. Experimental results showed that the quenching mechanism was a static quenching and there was one independent class of binding site on HIgG for SMZ during their interaction. The thermodynamic parameters of the reaction, namely standard enthalpy ΔH⁰ and entropy ΔS⁰, had been calculated to be ?19.12 kJ · mol^?1 and 20.22 J · mol^?1 · K^?1, respectively, which meant that the electrostatic interaction was the predominant intermolecular force in stabilizing the SMZ ? HIgG complex. Moreover, the conformational changes of HIgG in the presence of SMZ were confirmed by three‐dimensional fluorescence spectroscopy, UV–vis absorption spectroscopy and CD spectroscopy. Copyright © 2014 John Wiley & Sons, Ltd.     

Luminescence of bacteriorhodopsin from Halobacterium halobium and its connection with the photochemical conversions of the chromophore

Red luminescence of purple membranes from Halobacterium halobium cells in suspension, dry film or freeze-dried preparations was studied and its emission, excitation and polarization spectra are reported. The emission spectra have three bands at 665–670, 720–730 and at 780–790 nm. The position (maximum at 580 nm) and shape of the excitation spectra are close to those of the absorption spectra. The spectra depend on experimental conditions, in particular on pH of the medium. Acidification increases the long wavelength part of the emission spectra and shifts the main excitation maximum 50–60 nm to the longer wavelength side. Low-temperature light-induced changes of the absorption, emission and excitation spectra are presented. Several absorbing and emitting species of bacteriorhodopsin are responsible for the observed spectral changes. The bacteriorhodopsin photoconversion rate constant was estimated to be about 1 · 10¹¹ s^?1 at ? 196°C from the quantum yields of the luminescence (1 · 10^?3) and photoreaction (1 · 10^?1). The temperature dependence of the luminescence quantum yield points to the existence of two or three quenching processes with different activation energies. High degree of luminescence polarization (about 45–47%) throughout the absorption and fluorescence spectra and its temperature independence show that there is no energy transfer between bacteriorhodopsin molecules and no chromophore rotation during the excitation lifetime. In carotenoid-containing membranes, energy migration from the bulk of carotenoids to bacteriorhodopsin was not found either. Bacteriorhodopsin phosphorescence was not observed in the 500–1100 nm region and the emission is believed to be fluorescence by nature.     

Phosphorylation of Frog Photoreceptor Membranes induced by Light

LIGHT induces changes in the visual pigment rhodopsin and its retinaldehyde prosthetic group¹ but the specific chemical events which lead to photoreceptor excitation remain obscure. We describe here a phosphorylation reaction initiated by rhodopsin bleaching which may be part of the mechanism linking photon absorption to changes in membrane resistance.     

Squid nerve Na+/Ca2+ exchanger expressed in Saccharomyces cerevisiae: Up-regulation by a phosphorylated cytosolic protein (ReP1–NCXSQ) is identical to that of native exchanger in situ

This work shows, for the first time, a properly metabolically regulated squid nerve Na⁺/Ca²⁺ exchanger (NCXSQ1) heterologous expressed in Saccharomyces cerevisiae. The exchanger was fused to the enhanced green fluorescence protein (eGFP) on its C-terminus and had two tags, a Strep-tag II and 6 histidines, added to the N-terminal region (ST–6HB–NCXSQ1–eGFP). The eGFP fluorescence signal co-localized with that of the plasma membrane marker FM1-43 in whole cells that displayed an uptake of Ca²⁺ with the expected characteristics of the reverse Na⁺/Ca²⁺ exchange mode. Similar to squid nerve membrane vesicles, inside-out yeast plasma membrane vesicles (ISOV) showed a Ca²⁺_i regulation of the forward mode that was modulated by previously phosphorylated regulatory cytosolic protein (ReP1–NCXSQ). On the other hand, a close association between NCXSQ1 and ReP1–NCXSQ, estimated by co-immunoprecipitation, was independent of ReP1–NCXSQ phosphorylation. An additional crucial observation was that in proteoliposomes containing only the ST–6HB–NCXSQ1–eGFP protein, Na⁺/Ca²⁺ exchange was stimulated by phosphorylated ReP1–NCXSQ; i.e., this up-regulation needs no other requirement besides the lipid membrane and the exchanger protein. Finally, this work provides a potential approach to obtain enough purified NCXSQ1 for structural and biochemical studies which have been delayed due to the lack of sufficient material.     

Preparation and luminescence properties of KLaSiO4:Tb3+ phosphors

KLaSiO₄:Tb³⁺ phosphors were synthesized using the sol–gel method. The structure and luminescence properties of the materials were characterized using X-ray diffraction, Fourier transform infrared (FTIR) spectroscopy, thermogravimetry–differential thermal analysis, fluorescence spectra and calculated Commission Internationale de l'éclairage coordinates. The results showed that the material had a hexagonal structure, and that doping of Tb³⁺ did not change the crystal structure of KLaSiO₄. FTIR spectroscopy confirmed the existence of stretching vibrations of Si–O, bending vibrations of Si–O–Si, and asymmetric tensile vibrations of Si–O in KLaSiO₄. The excitation spectrum of the sample consisted of 4f⁷→5d¹ broadband absorption and the characteristic excitation peak of Tb³⁺, the excitation peak at 232 nm belongs to the spin allowed ⁷F_J→⁷D_J transition of Tb³⁺, the excitation peak at 268 nm belongs to the spin forbidden ⁷F_J→⁹D_J transition of Tb³⁺, and the absorption band of ⁷F_J→⁷D_J transition is split. Under excitation at 232 nm, the emission peak of the sample was composed of the ⁵D₄→⁷F_J (J = 6, 5, 4, 3) energy level transition of Tb³⁺. The highest emission peak is located at 543 nm, which belongs to the ⁵D₄→⁷F₅ transition and emits green light. Concentration quenching occurred when the Tb³⁺ doping concentration was greater than 1% mol, the quenching mechanism was an electric dipole–electric dipole action. When the ratio of citric acid to total metal ions was 1:1 and the annealing temperature was 800°C, the surface defects of the phosphors were greatly reduced, the quenching effect was reduced, and the luminous intensity reached the maximum.     

Molecular dynamics simulation of d-Benzedrine transmitting through molecular channels within D3R

Dex-Benzedrine (known as d-Benzedrine or SAT) acts in dopamine receptors of central nerve cell system. In clinic, SAT is used to treat a variety of diseases; meanwhile, it has dependence and addiction. In order to investigate the pharmacology and addiction mechanisms of SAT as a medicine, in this paper, we have studied the structure of D₃R complex protein with SAT, and based on which, using potential mean force with umbrella samplings and the simulated phospholipid bilayer membrane (or POPC bilayer membrane), the molecular dynamics simulation was performed to obtain free energy changes upon the trajectories for SAT moving along the molecular channels within D₃R. The free energy change for SAT transmitting toward the outside of cell along the functional molecular channel within D₃R is 83.5 kJ mol^?1. The change of free energy for SAT to permeate into the POPC bilayer membrane along the protective molecular channel within D₃R is 87.7 kJ mol^?1. Our previous work gave that the free energy for Levo-Benzedrine (RAT) transmitting toward the outside of cell along the functional molecular channel within D₃R is 91.4 kJ mol^?1, while it is 117.7 kJ mol^?1 for RAT to permeate into the POPC bilayer membrane along the protective molecular channel within D₃R. The values of free energy suggest that SAT relatively prefers likely to pass through the functional molecular channel within D₃R for increasing the release of dopamine molecules resulting in a variety of functional effects for SAT. The obtained results show that the pharmacology and addiction mechanisms of SAT as a drug are closely related to the molecular dynamics and mechanism for SAT transmitting along molecular channels within D₃R.     

The effect of thylakoid membrane reorganisation on chlorophyll fluorescence lifetime components: A comparison between state transitions,protein phosphorylation and the absence of Mg2+

Room temperature chlorophyll fluorescence lifetime measurements using single photon counting and low-intensity laser excitation have been carried out on photosynthetic systems which have undergone protein reorganisation by an in vivo state 1-state 2 transition, protein phosphorylation and the absence of Mg²⁺. Analysis of the global changes in average lifetime and total fluorescence yield suggest that each treatment brings about a decrease in Photosystem (PS) II absorption cross-section but that this mechanism of energy redistribution accounts for different proportions of the total fluorescence quenching in the various cases. Further analysis of the overall fluorescence decay into individual kinetic components was carried out using a four-exponential model. The state transition did not alter the lifetimes of the four components but decreased the fluorescence yield of the long-lived decay, at both F₀ and F_M, by 24% and increased the yield of the rapid components. Such changes infer that there is a decrease in PS II absorption cross-section and an increase in PS I excitation on going from state 1 to state 2. Furthermore, these alterations show that the 500 ps component (at F₀) gives rise to the 2 ns decay (at F_M). After in vitro protein phosphorylation at 5 mM Mg²⁺, the changes are very similar to those brought abought by a state transition, except that both long-lived kinetic components exhibit a decrease in yield. When protein phosphorylation was carried out at 2 mM Mg²⁺ a slight decrease in the lifetimes of the two slow components was observed, with a further decrease in the yield of the 2.3 ns decay and a larger increase in the yields of the two rapid decays. Although the fluorescence quenching brought about by the absence of Mg²⁺ (57%) was the largest of all the treatments, only a small part could be explained by a decrease in PS II absorption cross-section (17%). The absence of Mg²⁺ led to a decrease in the lifetimes and yields of the two long-lived decays. A careful comparison of the characteristics of the slowest component in the presence and absence of 5 mM Mg²⁺ on closing the PS II traps suggest that this decay has different origins in the two cases.     

Reduction kinetics of the photo-oxidized chlorophyll a+II in the nanosecond range: Measurements of the absorption changes at 688 nm under repetitive flash excitation

The 688 nm absorption changes (ΔA₆₈₈), indicating the photochemical turnover of chlorophyll a_II (Chl a_II) have been investigated under repetitive laser flash excitation conditions in spinach chlorplasts. It was found that under steady state conditions about 50–60% of the photo-oxidized primary donor of Photosystem II (PS II), Chl a⁺_II, becomes re-reduced with a biphasic kinetics in the nanosecond time scale with half-life times of about 50 ns and 400 ns. The remaining Chl a⁺_II becomes re-reduced in the microsecond range.     

Na+/H+ exchanger (NHE) in the basolateral membrane is encoded by NHE-1 mRNA in LLC-PK1 clone 4 cells

Several isoforms of Na⁺/H⁺ exchanger (NHE-1–5) have been identified. LLC-PK1 clone 4 (CL4) expresses the amiloride-sensitive type of NHE predominantly in the basolateral membrane, which is believed to be NHE-1. It is not clear whether CL4 expresses NHE in the apical membrane and which side of NHE is encoded by the NHE-1 mRNA. Using acidified CL4 cells on the filter membrane, we examined Na⁺-dependent pH recovery of the apical and basolateral membranes separately. Na⁺ applied to the apical membrane recovered cell pH. Na⁺-dependent pH recovery in the apical membrane was not inhibited by SITS, DIDS, or contralateral amiloride. Li⁺ but not K⁺, chol⁺, or NMG⁺ could replace Na⁺. These data are consistent with the presence of NHE in the apical membrane. Transfection with an antisense oligonucleotide corresponding to the 5′ terminal site of NHE-1 cDNA of CL4 decreased NHE activity in the basolateral membrane but not in the apical membrane. We conclude that CL4 expresses NHE activities in both apical and basolateralmembranes and that NHE-1 mRNA encodes NHE only in the basolateral membrane. J. Cell. Physiol. 171:318–324, 1997. © 1997 Wiley-Liss, Inc.     

Infrared spectra of outer and cytoplasmic membranes of Escherichia coli

Outer and cytoplasmic membranes of Escherichia coli were prepared by a method based on isopyenic centrifugation on a sucrose gradient. The infrared spectra of solid films of these membranes were studied. The cytoplasmic membrane had an amide I band at 1657 cm^?1 and an amide II band at 1548 cm^?1. The outer membrane had a broad amide I band at 1631–1657 cm^?1 and an amid II band at 1548 cm^?1 with a shoulder at 1520–1530 cm^?1. Upon deuteration, the amide I band of the cytoplasmic membrane shifted to 1648 cm^?1, whereas the band at 1631 cm^?1 of the outer membrane remained unchanged. After extraction of lipids with chloroform and methanol, the infrared spectra in the amide I and amide II regions of both membranes remained unchanged. Although the outer membrane specifically contained lipopolysaccharide, this could not account for the difference in the infrared spectra of outer and cytoplasmic membranes. It is concluded that a large portion of proteins in the outer membrane is a β-structured polypeptide, while this conformation is found less, if at all in the cytoplasmic membrane.     

Analysis of the interaction of gallic acid and myoglobin by UV-vis absorption spectroscopy

UV-vis absorption spectroscopy has been used to analyze the interaction of myoglobin (Мb) and gallic acid (GA). The binding constants (4.38 × 10⁴ M^–1 at 298.15 K and 0.42 × 10⁴ М^–1 at 308.15K), the number of binding sites (h = 1.0), and the thermodynamic parameters of binding (ΔH, ΔS, and ΔG) have been determined. Hydrogen bonds have been shown to play a major role in the stabilization of the GA–Мb complexes. GA binding led to slight changes in the electronic state of the heme ring of the protein.     

Isolation and Characterization of the Thylakoid Membranes from the NaCl-Resistant (NaClr) Mutant Strain of the Cyanobacterium Anabaena variabilis

NaCl-induced changes in the thylakoid membrane of wild-type Anabaena variabilis and its NaCl^r mutant strain have been studied. Biochemical characterization of the thylakoid membrane was done by taking its absorption and fluorescence spectra at different wavelength. The thylakoid membranes of both strains were isolated by mechanical disruption of the freeze-dried and lysozyme-treated cells, followed by differential and density gradient centrifugation. The light absorption spectra of the thylakoid membrane showed three and two peaks in NaCl^r mutant strain and its wild-type counterpart respectively at wavelengths of 400–850 nm. These peaks revealed that the thylakoid membrane contains a large amount of carotenoid and chlorophyll a. Fluorescence emission spectra of thylakoid membrane of NaCl^r mutant and its wild-type strain at excitation wavelength of 335 nm showed two different peaks, one at 340 nm and the other at 663 nm respectively. The light absorption and fluorescence spectra of the thylakoid membrane also revealed that the membrane contained carotenoid pigment, chlorophyll (Chl) a, and a pigment with an emission peak at 335 nm. The HPLC analysis of the pigments of the thylakoid membrane indicates that the NaCl^r mutant strain under NaCl stress contained an additional peak for the carotenoid pigment, which was lacking in its wild-type counterpart. The major peak in thylakoid membrane was that of echinenone and β-carotene. Whereas the polypeptide composition of thylakoid membrane differed in the wild-type and its NaCl^r mutant strain, no difference in the cell wall protein pattern was observed in both strains. The thylakoid membrane of NaCl^r mutant strain contained two additional protein bands that were absent in its wild-type counterpart. The thylakoid membrane of the wild-type and its NaCl^r mutant strain also showed morphological variations under NaCl stress. Received: 14 April 2000 / Accepted: 23 May 2000     

Ultrasonic absorption studies of poly-benzyl-L-aspartate in mixed solvents, in relation to the helix-cell transition

Ultrasonic absorption measurements were carried out on solutions of polybenzyl-L -aspartate (PBLA) in chloroform–dichloroacetic acid (DCA) and in 1,2-dichloroethane (DCE)–DCA, in the range 3.9–155 MHZ . The helix–coil transition of PBLA produces an increase of absorption which is larger in CHCl₃–DCA than in DCE–DCA solutions. The influence of the solvent on the excess ultrasonic absorption suggests that solvation processes may be involved in these changes of absorption. The plots of the absorption vs. the volume fraction of DCA do not show any absorption maximum. This indicates that the ultrasonic absorption is not sensitive to the helix–coil equilibrium of PBLA in the frequency range investigated. A maximum value of 10⁹ S ^?1 has been obtained for the rate constant of growth of a helix region.     

Formation of a Piperidinium Derivative from N-(5′-Chloropentyl)-N-methylaminoaceto-2,6-xylidide in relation to the Sustained Local Anaesthetic Action on the Sciatic Nerve of the Guinea-pig <Emphasis Type="Italic">in vivo</Emphasis>

THE possibility of using the intramolecular alkylation reaction of tertiary ω-haloalkylamines (Fig. 1) to overcome the cell membrane barrier to cyclic quaternary ammonium compounds seems to have been almost entirely overlooked. Some pharmacological applications of this principle have already been studied^1–3 and we feel that the possibility of obtaining local anaesthetic agents⁴ may be of therapeutic interest. The nerve sheaths and membranes hinder the penetration of quaternary ammonium compounds⁵, but tertiary amines in their base form are able to pass⁶. Thus, a tertiary haloalkylamine (I in Fig. 1) should be able to cross the barriers, and, because of its capacity to cyclize, the corresponding cyclic quaternary ammonium derivative (II) would be expected to be formed at least to some extent at the sites for blockade of nerve excitation. The barriers to outward diffusion of the quaternary compound formed are probably the same as for the inward movement, so that very slow disappearance can be expected. Accordingly, if the quaternary derivative has a local anaesthetic action, this would probably be of long duration. Our observation that tertiary haloalkylamine derivatives closely related to lidocaine have a long term anaesthetic effect⁴ supports this hypothesis. It would be valuable, however, to try to relate the local Q XCH3 JH3 -NHCOCH2N(CH2)5CI VCH3 I 'CH3 jJrX . -NHC0CH2N 4-Ct CH3 *? II anaesthetic effect in vivo with the amount of (I) and of the piperidinium derivative (II) formed in the nerves. One compound, N-(5 '-chloropentyl)-N-methylaminoaceto-2,6-xyli-dide (I in Fig. 1), tritium-labelled in the 1'-position (specific activity 10 mCi/mmol), was therefore selected for the following experiments.