Multifunctional liposomes for nasal delivery of the anti-Alzheimer drug tacrine hydrochloride

The purpose of this study was the development of multifunctional liposomes for nasal administration of tacrine hydrochloride. Liposomes were prepared using traditional excipients (cholesterol and phosphatidylcholine), partly enriched with α-tocopherol and/or Omega3 fatty acids. This approach was chosen in order to obtain at the same time two positive results: an enhanced drug permeation through nasal mucosa and a concomitant neuroprotective effect. Several liposome formulations were prepared using the Reverse Phase Evaporation technique followed by membrane filter extrusion. In particular, liposome capacity to enhance drug permeation was evaluated by means of membrane permeation and cellular uptake studies. Furthermore, liposome effect on neuronal viability and intracellular ROS production was evaluated as well as their cytoprotective effect against oxidative stress. All liposome formulations showed a mean diameter in the range of 175?nm to 219?nm with polydispersity index lower than 0.22, a lightly negative zeta potential and excellent encapsulation efficiency. Moreover, along with good mucoadhesive properties, multifunctional liposomes showed a markedly increase in tacrine permeability, which can be related to liposome fusion with cellular membrane, a hypothesis, which was also supported by cellular uptake studies. Finally, the addition of α-tocopherol without Omega3 fatty acids, was found to increase the neuroprotective activity and antioxidant properties of liposomes.     

Biochemical similarity among serologically distinct flagellins of Campylobacter jejuni

Abstract We describe a simplified method for obtaining highly purified flagellin, suitable for biochemical analysis using HPLC-gel permeation. Amino acid composition and N-terminal sequence analyses were performed on flagellins from serologically distinct isolates. The amino acid composition of flagellin from 10 strains was very similar. The N-terminal amino acid sequence is highly conserved. Significant sequence homology was found with flagellin of Bacillus subtilis .     

Induction of acetoacetate decarboxylase in Clostridium acetobutylicum

Abstract Factors that may initiate the biosynthesis of acetoacetate decarboxylase were investigated in resting cells of Clostridium acetobutylicum . Linear acids from C₁ to C₄ were inducers, whereas branched acids and linear acids from C₅ to C₇ were not inducers of acetoacetate decarboxylase biosynthesis. Induction of acetoacetate decarboxylase was maximal at pH 4.8 in the presence of acid concentrations comparable with those found during fermentation. In growth conditions repression of acetoacetate decarboxylase biosynthesis was found. This fact explains that acetone production by Clostridium acetobutylicum occurs when growth slows down.     

Growing a stratified,cornified primary culture of rat keratinocytes with epidermis-like water permeation barrier function

Summary The culture of cutaneous keratinocytes grown on a Puropore nylon microporous membrane at the air-liquid interface has been shown to be similar to the epidermis in a number of molecular and morphologic characteristics but to exhibit a significantly greater degree of tritiated water permeation. Various culture conditions have been altered in an effort to improve the water barrier properties. A Kp value in the range of 5.5±1.6×10⁻³ has been obtained for 79% of the culturea) by plating 0.9×10⁶ viable basal cells on a piece (13-mm diameter) of membrane for 7 days of submerged growth,b) by placing two membranes on two stacked glass fiber filters (47-mm extra-thick) in a culture dish (60 mm) for 14 days of growth at the air-liquid interface,c) by replacing the growth medium, i.e., 1 ml of complete minimum essential medium (CMEM) every 24 h after lifting,d) by using 10% fetal bovine serum (FBS) in the CMEM during the submerged culture period and 15% FBS in the CMEM during the lifted culture period, ande) by adding a dialysis membrane on top and a Puropore nylon membrane below the culture when the cultures were inserted in the permeation cell for testing. The percentage of cultures with this value for Kp can be increased to 90% if only cultures with yellow, smooth, and shiny surfaces are tested. This system should be useful as a replacement for skin in testing the cutaneous permeation of some chemicals. To whom correspondence should be addressed at 1528 Public Health, The University of Michigan, Ann Arbor, Michigan 48109-2029.     

Halide permeation through three types of epithelial anion channels after reconstitution into giant liposomes

Anion-selective channels from apical membranes of cultured CFPAC-1 cells were isolated and incorporated into giant liposomes for patch clamp recording. Liposomes were formed from L--lecithin by a dehydration-hydration method. Ion channels were characterized using the excised inside-out patch clamp configuration. The most commonly observed anion channels were similar to those observed in native epithelial tissues. The linear 20 pS Cl channel had the halide permeability sequence Cl^– > I^– Br^– > F^–, and showed anomalous mole-fraction behavior in solutions containing different proportions of Cl^– and F^–, ions. The autwardly rectifying Cl^– channel had the halide permeability sequence I^– > Br^– > Cl^– > F^–, and also showed anomalous molefraction behavior, indicating that both these channels probably contain multi-ion pores. The third, voltage-dependent anion channel showed at least five different substrates, had a conductance of 390 pS in the main state, and showed two types of kinetics, fast (openings and closings < 1 ms), and slow (openings and closings > 1 s). The channel was seen more frequently after reconstitution into giant liposomes than in intact cells. It was not selective amongst the halides, and there was no deviation from a linear dependence of relative current on molar fractions, indicating relatively simple permeation through the pore. Differences in halide permeabilities suggest that different anion channels may be related to different membrane proteins. Comparison with the chloride channel proteins isolated biochemically from epithelial cell membranes is discussed. Correspondence to: M. Duszyk     

A site accessible to extracellular TEA+ and K+ influences intracellular Mg2+ block of cloned potassium channels

The members of the RCK family of cloned voltage-dependent K⁺ channels are quite homologous in primary structure, but they are highly diverse in functional properties. RCK4 channels differ from RCK1 and RCK2 channels in inactivation and permeation properties, the sensitivity to external TEA, and to current modulation by external K⁺ ions. Here we show several other interesting differences: While RCK1 and RCK2 are blocked in a voltage and concentration dependent manner by internal Mg²⁺ ions, RCK4 is only weakly blocked at very high potentials. The single-channel current-voltage relations of RCK4 are rather linear while RCK2 exhibits an inwardly rectifying single-channel current in symmetrical K⁺ solutions. The deactivation of the channels, measured by tail current protocols, is faster in RCK4 by a factor of two compared with RCK2. In a search for the structural motif responsible for these differences, point mutants creating homology between RCK2 and RCK4 in the pore region were tested. The single-point mutant K533Y in the background of RCK4 conferred the properties of Mg²⁺ block, tail current kinetics, and inward ion permeation of RCK2 to RCK4. This mutant was previously shown to be responsible for the alterations in external TEA sensitivity and channel regulation by external K⁺ ions. Thus, this residue is expected to be located at the external side of the pore entrance. The data are consistent with the idea that the mutation alters the channel occupancy by K⁺ and thereby indirectly affects internal Mg²⁺ block and channel closing.Abbreviations TEA tetraethylammonium - EGTA Ethylene glycol-bis (-aminoethyl ether) N,N,N,N-tetraacetic acid - 2S3B model 2-site 3-barrier model Correspondence to: S. H. Heinemann     

Thermodynamically specific gating kinetics of cardiac mammalian K (ATP) + channels in a physiological environment near 37°C

Elementary K⁺ currents through isolated ATP-sensitive K⁺ channels from neonatal rat cardiocytes were recorded to study their temperature dependence between 9°C and 39°C. Elementary current size and, thus, K⁺ permeation through the open pore varied monotonically with temperature with a Q₁₀ of 1.25 corresponding to a low activation energy of 3.9 kcal/mol. Open-state kinetics showed a complicated temperature dependence with Q₁₀ values of up to 2.94. Arrhenius anomalies of _open(1) and _open(2) indicate the occurrence of thermallyinduced perturbations with a dominating influence on channel portions that are involved in gating but are obviously ineffective in altering pore-forming segments. At 39°C, open-state exit reactions were associated with the highest activation energy (O₂ exit reaction: 12.1 kcal/ mol) and the largest amount of entropy. A transition from 19°C to 9°C elucidated a paradoxical kinetic response, shortening of both O-states, irrespective of the absence or presence of cAMP-dependent phosphorylation. Another member of the K⁺ channel family and also a constituent of neonatal rat cardiocyte membranes, 66 pS outwardly-rectifying channels, was found to react predictably since _open increased on cooling. Obviously, cardiac K _(ATP) ⁺ channels do not share this exceptional kinetic responsiveness to a temperature transition from 19°C to 9°C with other K⁺ channels and have a unique sensitivity to thermally-induced perturbations.     

Permeation of Ca2+ through K+ channels in the plasma membrane of Vicia faba guard cells

Summary The whole-cell patch-clamp method has been used to measure Ca²⁺ influx through otherwise K⁺-selective channels in the plasma membrane surrounding protoplasts from guard cells of Vicia faba. These channels are activated by membrane hyperpolarization. The resulting K⁺ influx contributes to the increase in guard cell turgor which causes stomatal opening during the regulation of leaf-air gas exchange. We find that after opening the K⁺ channels by hyperpolarization, depolarization of the membrane results in tail current at voltages where there is no electrochemical force to drive K⁺ inward through the channels. Tail current remains when the reversal potential for permeant ions other than Ca²⁺ is more negative than or equal to the K⁺ equilibrium potential (–47 mV), indicating that the current is due to Ca²⁺ influx through the K⁺ channels prior to their closure. Decreasing internal [Ca²⁺] (Ca _i ) from 200 to 2 nm or increasing the external [Ca²⁺] (Ca _o ) from 1 to 10 mm increases the amplitude of tail current and shifts the observed reversal potential to more positive values. Such increases in the electrochemical force driving Ca²⁺ influx also decrease the amplitude of time-activated current, indicating that Ca²⁺ permeation is slower than K⁺ permeation, and so causes a partial block. Increasing Ca _o also (i) causes a positive shift in the voltage dependence of current, presumably by decreasing the membrane surface potential, and (ii) results in a U-shaped current-voltage relationship with peak inward current ca. –160 mV, indicating that the Ca^2– block is voltage dependent and suggesting that the cation binding site is within the electric field of the membrane. K⁺ channels in Zea mays guard cells also appear to have a Ca _i -, and Ca _o -dependent ability to mediate Ca²⁺ influx. We suggest that the inwardly rectiying K⁺ channels are part of a regulatory mechanism for Ca _i . Changes in Ca _o and (associated) changes in Ca _i regulate a variety of intracellular processes and ion fluxes, including the K⁺ and anion fluxes associated with stomatal aperture change.This work was supported by grants to S.M.A. from NSF (DCB-8904041) and from the McKnight Foundation. K.F.-G. is a Charles Gilbert Heydon Travelling Fellow. The authors thank Dr. R. MacKinnon (Harvard Medical School) and two anonymous reviewers for helpful comments.     

Auxin-induced changes in the cell wall structure: Changes in the sugar compositions, intrinsic viscosity and molecular weight distributions of matrix polysaccharides of the epicotyl cell wall of Vigna angularis

Rapid effects of indole-3-acetic acid (IAA) on the mechanical properties of cell wall, and sugar compositions, intrinsic viscosity and molecular weight distribution of cell wall polysaccharides were investigated with excised epicotyl segments of Vigna angularis Ohwi et Ohashi cv. Takara.

• 1 IAA caused cell wall loosening as studied by stress-relaxation analysis within 15 min after the IAA application.
• 2 IAA stimulated the decrease in the content of arabinose and galactose in the hemicellulose 1 h after its application. The amounts of other component sugars in the cell wall polysaccharides remained constant during the IAA-induced segment growth.
• 3 The intrinsic viscocity of the pectin increased as early as 30 min after the IAA application. This effect was not prevented when elongation growth of the segment was osmotically suppressed by 0.15 M mannitol.
• 4 Gel permeation chromatography of the pectin on a Sepharose 4 B column demonstrated that IAA caused increase in the mass-average molecular weight of the pectin. Analysis of the sugar compositions of the pectin eluted from the Sepharose 4 B column indicated that IAA increased the molecular weight of the polysaccharides composed of uronic acid, galactose, rhamnose and arabinose. This effect became apparent within 30 min after the IAA application. Furthermore, IAA increased the molecular weight of the pectin when elongation growth of the epicotyl segments was osmotically suppressed by 0.15 M mannitol.
• 5 Hemicellulose of the cell wall chromatographed on a Sepharose CL-4 B column. Analysis of the neutral sugar compositions and the iodine staining property (specific for xyloglucans) of the polysaccharide solution eluted from the column indicated that the hemicellulose consisted of xyloglucans, arabinogalactans and polysaccharides composed of xylose and/or mannose. IAA caused a decrease in the arabinogalactan content and depolymerization of xyloglucans. These IAA effects became apparent within 30 min after the IAA application. These changes occurred even when elongation growth of the epicotyl segments was osmotically suppressed by 0.15 M mannitol.

Polymerization of the pectin, degradation of arabinogalactans and depolymerization of xyloglucans appear to be involved in the mechanism by which IAA induces cell wall loosening and therefore extension growth of cells.     

A Refined Open State of the Glycine Receptor Obtained via Molecular Dynamics Simulations

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