Interactions of valproic acid with lipid membranes of 1,2-dimyristoyl-sn-glycero-3-phosphocholine.

Lipid bilayers of 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) were prepared in two forms, as a suspension of multilamellar spherical vesicles and as planar membranes deposited on a conductive solid support. We used Fourier Transformed Infrared (FTIR) and Raman spectroscopic techniques to study the lipid vesicles while the solid supported bilayers were characterized by using electrochemical experiments (cyclic voltammetry and impedance). Valproic acid (Valp) was either present in the solution or incorporated into the lipid structure. As the Valp:DMPC ratio increases the phase transition temperature decreases while the phase transition becomes less marked. Moreover, for the Valp:DMPC complex species a slight decrease in the number of gauche isomers was observed relative to the number of trans isomers what corresponds to an increase in the packing density of the acylic chains. Based on derived electrical properties of the supported membranes it can be concluded that Valp induces the formation of pores and other defects in the lipid films. Valp incorporated into the membrane is seriously detrimental to the bilayer stability.

Characterization of interactions of eggPC lipid structures with different biomolecules.

In this paper we study the interactions of two biomolecules (ascorbic acid and Annonacin) with a bilayer lipid membrane. Egg yolk phosphatidylcholine (eggPC) liposomes (in crystalline liquid state) were prepared in solutions of ascorbic acid (AA) at different concentration levels. On the other hand, liposomes were doped with Annonacin (Ann), a mono-tetrahydrofuran acetogenin (ACG), which is an effective citotoxic substance. While AA pharmacologic effect and action mechanisms are widely known, those of Ann's are only very recently being studied. Both Fourier Transformed Infrared (FTIR) and Raman spectroscopic techniques were used to study the participation of the main functional groups of the lipid bilayer involved in the membrane-solution interaction. The obtained spectra were comparatively analyzed, studying the spectral bands corresponding to both the hydrophobic and the hydrophilic regions in the lipid bilayer. Electrochemical experiments namely; impedance spectroscopy (EIS) and cyclic voltamperometry (CV) were used as the main characterization techniques to analyse stability and structural changes of a model system of supported EggPC bilayer in connection with its interactions with AA and Ann. At high molar ratios of AA, there is dehydration in both populations of the carbonyl group of the polar head of the lipid. On the other hand, Ann promotes the formation of hydrogen bonds with the carbonyl groups. No interaction between AA and phosphate groups is observed at low and intermediate molar ratios. Ann is expected to be able to induce the dehydration of the phosphate groups without the subsequent formation of H bonds with them. According to the electrochemical analysis, the interaction of AA with the supported lipid membrane does not alter its dielectric properties. This fact can be related to the conservation of structured water of the phosphate groups in the polar heads of the lipid. On the other hand, the incorporation of Ann into the lipid membrane generates an increase in the number of defects while changes the dielectric constant. This, in turn, can be associated with the induced dehydration of the phosphate groups.

Oligothiophene wires: impact of torsional conformation on the electronic structure.

Charge transport in polymer- and oligomer-based semiconductor materials depends strongly on the structural ordering of the constituent molecules. Variations in molecular conformations influence the electronic structures of polymers and oligomers, and thus impact their charge-transport properties. In this study, we used Scanning Tunneling Microscopy and Spectroscopy (STM/STS) to investigate the electronic structures of different alkyl-substituted oligothiophenes displaying varied torsional conformations on the Au(111) surface. STM imaging showed that on Au(111), oligothiophenes self-assemble into chain-like structures, binding to each other via interdigitated alkyl ligands. The molecules adopted distinct planar conformations with alkyl ligands forming cis- or trans- mutual orientations. For each molecule, by using STS mapping, we identify a progression of particle-in-a-box-like states corresponding to the LUMO, LUMO+1 and LUMO+2 orbitals. Analysis of STS data revealed very similar unoccupied molecular orbital energies for different possible molecular conformations. By using density functional theory calculations, we show that the lack of variation in molecular orbital energies among the different oligothiophene conformers implies that the effect of the Au-oligothiophene interaction on molecular orbital energies is nearly identical for all studied torsional conformations. Our results suggest that cis-trans torsional disorder may not be a significant source of electronic disorder and charge carrier trapping in organic semiconductor devices based on oligothiophenes.

Estimating diffusion coefficients of probe molecules into polyelectrolyte brushes by electrochemical impedance spectroscopy.

Molecular transport through thin polymer films has become a subject with a variety of challenges and opportunities for chemists, physicists, and material scientists in recent years. The diffusion of probe molecules in and out of macromolecular environments plays a major role in the response of polymer-based sensor materials or the design of time-released drug delivery systems. Obtaining an improved understanding of the relevant dynamic phenomena, like transport of molecular probes, in boundary layers represents a crucial step to develop a clearer picture of the molecular transport processes taking place at interfaces modified with macromolecular assemblies. In this work, we present a new approach based on the derivation of the theoretical impedance transfer function to unambiguously describe the impedance response of gold electrodes modified with poly(methacryloyloxy)-ethyl-trimethyl-ammonium chloride (PMETAC) brushes. We demonstrate that this methodology not only enables the description of the experimental data but also provides insightful information about the dynamics of the diffusion of probe molecules inside the brush. More important, we show the capabilities of electrochemical impedance spectroscopy to gather information on a molecular transport process inside the brush under experimental conditions in which other electrochemical techniques are no longer applicable. As such, we consider that this experimental approach constitutes a new and powerful tool to estimate diffusion coefficients of probe molecules into interfacial macromolecular assemblies.

Dynamic regulation of middle neurofilament RNA pools during optic nerve regeneration.

Stereotypical changes in neurofilament subunit expression are highly correlated with the regenerative success of lower vertebrate CNS axons. The phylogenetically conserved binding of ribonucleoproteins to the 3'-untranslated region of the middle neurofilament subunit (NF-M) mRNA suggests that post-transcriptional mechanisms play an important role in the control of NF-M expression. To assess their contribution to the regulated changes in NF-M expression that occur during Xenopus laevis optic axon regeneration, we followed changes in intracellular NF-M RNA pools. Within 3 days after axotomy, when NF-M mRNA levels decrease in the injured retinal ganglion cells, heterogenous nuclear RNA levels increased more than 15-fold, but did so in both the operated and the contralateral unoperated eyes as compared with the eyes of surgically naive frogs. Increased nuclear RNA levels persisted throughout regeneration but never correlated directly with changes in mRNA expression, indicating that such changes most likely arose from alterations in nuclear-cytoplasmic RNA export and turnover. The early phase of optic nerve regeneration also exhibited an increase in the efficiency of translation of NF-M mRNA relative to surgically naive animals. This increase was only transient in unoperated control eye, but persisted through the peak of regeneration in the operated eye. Thus, post-transcriptional control of NF-M expression plays a significant role in regulating the cytoskeletal composition of injured neurons. These findings indicate that changes in protein expression during successful regeneration of CNS axons involve a complex interplay of transcriptional and translational regulation that is controlled by the operation of functional neuronal pathways. These findings also raise the additional possibility that factors regulating post-transcriptional changes in cytoskeletal gene expression may be as important as transcription factors for the successful regeneration of CNS axons.

Impedance analysis of ion transport through supported lipid membranes doped with ionophores: a new kinetic approach.

Kinetics of facilitated ion transport through planar bilayer membranes are normally analyzed by electrical conductance methods. The additional use of electrical relaxation techniques, such as voltage jump, is necessary to evaluate individual rate constants. Although electrochemical impedance spectroscopy is recognized as the most powerful of the available electric relaxation techniques, it has rarely been used in connection with these kinetic studies. According to the new approach presented in this work, three steps were followed. First, a kinetic model was proposed that has the distinct quality of being general, i.e., it properly describes both carrier and channel mechanisms of ion transport. Second, the state equations for steady-state and for impedance experiments were derived, exhibiting the input-output representation pertaining to the model's structure. With the application of a method based on the similarity transformation approach, it was possible to check that the proposed mechanism is distinguishable, i.e., no other model with a different structure exhibits the same input-output behavior for any input as the original. Additionally, the method allowed us to check whether the proposed model is globally identifiable (i.e., whether there is a single set of fit parameters for the model) when analyzed in terms of its impedance response. Thus, our model does not represent a theoretical interpretation of the experimental impedance but rather constitutes the prerequisite to select this type of experiment in order to obtain optimal kinetic identification of the system. Finally, impedance measurements were performed and the results were fitted to the proposed theoretical model in order to obtain the kinetic parameters of the system. The successful application of this approach is exemplified with results obtained for valinomycin-K(+) in lipid bilayers supported onto gold substrates, i.e., an arrangement capable of emulating biological membranes.

Impedance analysis of ion transport through gramicidin channels in supported lipid bilayers.

Selectivity between monovalent cations and its sequence of conductivity in lipid bilayers doped with the antibiotic Gramicidin D (GD) were examined using EIS. Experiments were performed using lipid bilayers obtained from a lipid mixture of phosphatidylcholine and dimethyldioctadecylammonium chloride (DODAC). Lipid bilayers were supported on gold surfaces modified with a mercapto-carboxylic acid. The bilayers were formed by chemisorption of this last species to form the first monolayer on gold and subsequent fusion of unilamellar vesicles to form an external bilayer attached by electrostatic interactions. A mathematical expression for the impedance of the membrane processes was derived. Some predictions of the presented model were checked after fitting the experimental results in various electrolyte compositions.

Structure, biological activity of the upstream regulatory sequence, and conserved domains of a middle molecular mass neurofilament gene of Xenopus laevis.

During development, the molecular compositions of neurofilaments (NFs) undergo progressive modifications that correlate with successive stages of axonal outgrowth. Because NFs are the most abundant component of the axonal cytoskeleton, understanding how these modifications are regulated is essential for knowing how axons control their structural properties during growth. In vertebrates ranging from lamprey to mammal, orthologs of the middle molecular mass NF protein (NF-M) share similar patterns of expression during axonal outgrowth, which suggests that these NF-M genes may share conserved regulatory elements. These elements might be identified by comparing the sequences and activities of regulatory domains among the vertebrate NF-M genes. The frog, Xenopus laevis, is a good choice for such studies, because its early neural development can be observed readily and because transgenic embryos can be made easily. To begin such studies, we isolated genomic clones of Xenopus NF-M(2), tested the activity of its upstream regulatory sequence (URS) in transgenic embryos, and then compared sequences of regulatory regions among vertebrate NF-M genes to search for conserved elements. Studies with reporter genes in transgenic embryos found that the 1. 5 kb URS lacked the elements sufficient for neuron-specific gene expression but identified conserved regions with basal regulatory activity. These studies further demonstrated that the NF-M 1.5 kb URS was highly susceptible to positional effects, a property that may be relevant to the highly variant, tissue-specific expression that is seen among members of the intermediate filament gene family. Non-coding regions of vertebrate NF-M genes contained several conserved elements. The region of highest conservation fell within the 3' untranslated region, a region that has been shown to regulate expression of another NF gene, NF-L. Transgenic Xenopus may thus prove useful for testing further the activity of conserved elements during axonal development and regeneration.

Xenopus laevis peripherin (XIF3) is expressed in radial glia and proliferating neural epithelial cells as well as in neurons.

Neuronal intermediate filament (nIF) proteins form the most abundant component of the axonal cytoskeleton. Thus, understanding their function and the regulation of their expression is essential for comprehending how axonal structure is regulated. Although most vertebrate nIF proteins are classified as type IV intermediate filament (IF) proteins, additional nIF proteins exist in frogs (Xenopus laevis), cyprinid fishes, and mammals (called XIF3, plasticin, and peripherin, respectively) that are classified as type III. Expression of a type III nIF protein is correlated strongly with the earliest phases of axonal outgrowth in fishes but less so in mammals. To understand better how the correlation between type III nIF protein expression and early phases of axonal outgrowth has changed during evolution, the authors examined XIF3 expression in Xenopus laevis. In Xenopus, the association between XIF3 expression and early axonal outgrowth was especially strong. For example, during early axonal development, XIF3 expression preceded and was more abundant and widespread than that of any of the type IV nIF proteins. As axons matured, neuronal expression of XIF3 gradually became more restricted while that of type IV nIF proteins increased. These results support the idea that type III nIF proteins play a special role during early phases of axonal outgrowth. In addition to finding XIF3 in neurons, the authors also unexpectedly found it in regions of the central nervous system that contain proliferating cells and radial glia. As a framework for interpreting variations in nIF expression in different vertebrate species, the authors built phylogenetic trees to clarify relationships among vertebrate nIF proteins. These trees supported the classification of XIF3, plasticin, and peripherin as orthologs (products of the same genetic locus, evolving separately only since the species lineages diverged). Thus, XIF3, plasticin, and peripherin probably should be referred to as Xenopus, fish, and mammalian peripherin, respectively. This finding argues that differences in expression of these three proteins in frogs, fishes, and mammals are the result of regulatory changes to the peripherin ancestral gene along each lineage. The expression of a peripherin ortholog in Xenopus glia may represent either an adaptation that arose since the divergence of Xenopus from mammals or, alternatively, a feature retained from an ancestral IF protein that was expressed originally both in neurons and in glia.

Sequence and expression patterns of two forms of the middle molecular weight neurofilament protein (NF-M) of Xenopus laevis.

The middle molecular weight neurofilament protein (NF-M) is relevant to our understanding of vertebrate neurofilaments in growing axons, both because it exists in all vertebrates and because it undergoes characteristic changes in its phosphorylation state during axonal development. Indeed, all vertebrate neurofilament proteins are believed to have originated by gene duplication from an ancestral, NF-M-like protein. The role of NF-M in axonal development has been studied extensively in the frog, Xenopus laevis, through the use of monoclonal antibodies. To acquire a better understanding of the relationship of X. laevis NF-M to that of other vertebrates and to obtain additional reagents to study and perturb neurofilaments in developing axons, we isolated cDNA clones from the nervous system. These clones encoded two forms of NF-M, which exhibited 93% amino acid identity overall and 94%, 96% and 90% identity over their head, rod, and tail domains, respectively. Synonymous nucleotide substitution rates between the two forms tied their origin to an ancestral duplication of the Xenopus genome, which occurred approximately 30 million years ago. Non-synonymous substitution rates indicated that the tail domain is evolving more rapidly than the rod domain. Both forms shared structural features in common with other vertebrate NF-Ms but had only a single example of the KSP phosphorylation motif that is repeated multiple times in the NF-Ms of bird, goldfish and mammal. In post-metamorphic frogs, the NF-M(1) transcript was consistently expressed at higher levels than that of NF-M(2), although their anatomical patterns of expression were qualitatively similar. During development, however, only NF-M(2) was detectable in retinal ganglion cells through stage 42. We speculate that the differences observed between these two forms may represent early stages of protein diversification akin to what occurred after the gene duplications that gave rise to other vertebrate neurofilament proteins.

The Xenopus laevis homologue to the neuronal cyclin-dependent kinase (cdk5) is expressed in embryos by gastrulation.

Phosphorylation of the neuronal cytoskeletal proteins NF-H, NF-M and tau is important for normal axonal development, and is involved in axonal injury and neurodegenerative diseases. In mammalian neurons, one kinase that phosphorylates these axonal cytoskeletal proteins is cyclin-dependent kinase 5 (cdk5). Cdk5 is a member of the family of cyclin-dependent kinases (cdks), whose other family members regulate mitosis. Unlike the other cdks, cdk5 is abundant in differentiated neurons. Embryos of the clawed frog Xenopus laevis have proved useful for studying other cyclin-dependent kinases, neurofilament proteins and tau during development. As a first step in studying the role of cdk5 and its effects on neurofilaments during Xenopus neural development, four cDNA clones were isolated by screening a frog brain cDNA library at lowered stringency with a cDNA probe to rat cdk5. The frog cdk5 clones encoded a protein of 292 amino acids that was 97% identical to rat cdk5. In situ hybridization demonstrated that the Xenopus cdk5 transcript, like that of mammals, was expressed in differentiated post-mitotic neurons. The high degree of sequence homology and shared neuronal expression suggests that the role of cdk5 in neurons is highly conserved between mammals and amphibians. Northern blot analysis indicated that during Xenopus development, cdk5 mRNA was first expressed between the midblastula transition and gastrulation, which both occur long before neuronal differentiation. These stages mark the transition from synchronous to asynchronous cell division and are the earliest stages of zygotic gene expression. This early expression of Xenopus cdk5 mRNA implies a role for cdk5 during embryogenesis that is separate from its role as an axonal cytoskeletal protein kinase. These observations provide the foundation for exploiting X. laevis embryos to study the role of cdk5 both in the early stages of axonal differentiation and also in early embryogenesis.