Autonomous transmembrane segment S4 of the voltage sensor domain partitions into the lipid membrane.

The S4 transmembrane segment in voltage-gated ion channels, a highly basic alpha helix, responds to changes in membrane potential and induces channel opening. Earlier work by others indicates that the S4 segment interacts with lipids in plasma membrane, but its mechanism is unclear. Working with synthetic tryptophan-labeled S4 peptides, we characterized binding of autonomous S4 to lipid membranes. The binding free energy (5.2 +/- 0.2 kcal/mol) of the peptide-lipid interaction was estimated from the apparent dissociation constants, determined from the changes in anisotropy of tryptophan fluorescence induced by addition of lipid vesicles with 30 mol% phosphatidylglycerol. The results are in good agreement with the prediction based on the Wimley-White hydrophobicity scale for interfacial (IF) binding of an alpha-helical peptide to the lipid bilayer (6.98 kcal/mol). High salt inhibited the interaction, thus indicating that the peptide/membrane interaction has both electrostatic and non-electrostatic components. Furthermore, the synthetic S4 corresponding to the Shaker potassium channel was found to spontaneously penetrate into the negatively charged lipid membrane to a depth of about 9 A. Our results revealed important biophysical parameters that influence the interaction of S4 with the membrane: they include fluidity, surface charge, and surface pressure of the membrane, and the at helicity and regular spacing of basic amino-acid residues in the S4 sequence.

Kinetic analysis of the interaction between poly(amidoamine) dendrimers and model lipid membranes.

We used fluorescence spectroscopy and surface tensiometry to study the interaction between low-generation (G1 and G4) poly(amidoamine) (PAMAM) dendrimers, potential vehicles for intracellular drug delivery, and model lipid bilayers. Membrane association of fluorescently labeled dendrimers, measured by fluorescence anisotropy, increased with increasing size of the dendrimer and with increasing negative charge density in the membrane, indicating the electrostatic nature of the interaction. When the membrane was doped with pyrene-labeled phosphatidyl glycerol (pyrene-PG), pyrene excimer fluorescence demonstrated a dendrimer-induced selective aggregation of negatively charged lipids when the membrane was in the liquid crystalline state. A nonlinear Stern-Volmer quenching of dendrimer fluorescence with cobalt bromide suggested a dendrimer-induced aggregation of lipid vesicles, which increased with the dendrimer's generation number. Surface tensiometry measurements showed that dendrimers penetrated into the lipid monolayer only at subphysiologic surface pressures (<30mN/m). We conclude that the low-generation PAMAM dendrimers associate with lipid membranes predominantly electrostatically, without significantly compromising the bilayer integrity. They bind stronger to membranes with higher fluidity and lower surface pressure, which are characteristic of rapidly dividing cells.

Impact of hybrid delivery of education on student academic performance and the student experience.

OBJECTIVES: To compare student academic performance and the student experience in the first-year doctor of pharmacy (PharmD) program between the main and newly opened satellite campuses of the University of Maryland. METHODS: Student performance indicators including graded assessments, course averages, cumulative first-year grade point average (GPA), and introductory pharmacy practice experience (IPPE) evaluations were analyzed retrospectively. Student experience indicators were obtained via an online survey instrument and included involvement in student organizations; time-budgeting practices; and stress levels and their perceived effect on performance. RESULTS: Graded assessments, course averages, GPA, and IPPE evaluations were indistinguishable between campuses. Students' time allocation was not different between campuses, except for time spent attending class and watching lecture videos. There was no difference between students' stress levels at each campus. CONCLUSIONS: The implementation of a satellite campus to expand pharmacy education yielded academic performance and student engagement comparable to those from traditional delivery methods.

A molecular dynamics model of the Bt toxin Cyt1A and its validation by resonance energy transfer.

Cyt1A is a cytolytic toxin from Bacillus thuringiensis var. israelensis. A computer model of the toxin in solution was generated and validated by resonance energy transfer (RET). The average distance between the two tryptophans (residues 158 and 161) and the fluorescently labeled cysteine 190 was 2.16 nm, which closely matched the distance predicted in computer simulations, 2.2 nm. The simulation results were able to explain two previous experimental observations: (i) amino-acid sequences of all Cyt toxins contain four blocks of highly conserved residues; and (ii) several single-point mutations drastically abrogated Cyt1A's toxicity. Selective randomization of atomic coordinates in the computer model revealed that the conserved blocks are important for proper folding and stability of the toxin molecule. Replacing lysine 225 with alanine, a mutation that renders the toxin inactive, was shown to result in breaking the hydrogen bonds between K225 and V126, L123, and Y189. Calculated Helmholtz free energy difference of the inactive mutation K225A was higher by 12 kcal/mol and 5 kcal/mol than the values for the benign mutations K118A and K198A, respectively, which indicates that the K225A mutant is significantly destabilized. The normal-mode and principal-component analyses revealed that in the wild-type Cyt1A the region around the residue K225 is quite stationary, due to the hydrogen-bond network around K225. In contrast, pronounced twisting and stretching were observed in the mutant K225A, and the region around the residue K225 becomes unstable. Our results indicate that conformational differences in this mutant spread far away from the site of the mutation, suggesting that the mutant is inactivated due to an overall change in conformation and diminished stability rather than due to a localized alteration of a "binding" or "active" site.

A fluorescence spectroscopy study on the interactions of the TAT-PTD peptide with model lipid membranes.

Protein-transduction domains (PTDs) have been shown to translocate into and through the living cells in a rapid manner by an as yet unknown mechanism. Regardless of the mechanism of translocation, the first necessary step must be binding of the PTD peptide to the surface of the lipid membrane. We used fluorescence spectroscopy to study the interaction between PTD of the HIV-1 Tat protein (TAT-PTD; residues 47-60 of Tat, fluorescently labeled with tryptophan) and the lipid bilayer labeled with various fluorescence membrane probes. The TAT-PTD tryptophan exhibited a decrease in fluorescence intensity and an increase in anisotropy upon interaction with lipid bilayers. The fluorescence changes were linearly proportional to the density of negative charge in the membrane. Kinetic analysis of the interaction showed two apparent dissociation constants. The value of one dissociation constant (Kd1 = 2.6 +/- 0.6 microM), which accounted for 24% of the interaction, was found to be independent of the negative charge density, suggesting its nonelectrostatic nature. The value of the second dissociation constant (Kd2), which accounted for 76% of the interaction, decreased linearly from 610 +/- 150 to 130 +/- 30 microM with an increase in negative charge density from 0 to 25 mol %, suggesting this interaction is electrostatic in nature. Even though the binding was predominantly electrostatic, it could not be reversed by high salt, indicating the presence of a second, irreversible, step in the interaction with lipid. When TAT-PTD was bound to lipid vesicles labeled with 1-(4-trimethylammoniumphenyl)-6-phenyl-1,3,5-hexatriene (TMA-DPH), fluorescence resonance energy transfer between the tryptophan and the probe occurred at a distance of 3.4 nm. No change in fluorescence anisotropy of either TMA-DPH or DPH was observed upon the interaction with TAT-PTD, indicating no significant disruption or perturbation of the lipid bilayer by the peptide. TAT-PTD did not cause dissipation of membrane potential (165 mV, negative inside). Inclusion of 3% pyrene-labeled phosphatidylglycerol (pyrene-PG) in the membrane revealed that TAT-PTD preferentially bound to the membrane in the liquid state. We conclude that membrane fluidity is an important physicochemical parameter, which may regulate binding of TAT-PTD to the membrane.

Amyloid-beta-induced pathological behaviors are suppressed by Ginkgo biloba extract EGb 761 and ginkgolides in transgenic Caenorhabditis elegans.

Amyloid-beta (Abeta) toxicity has been postulated to initiate synaptic loss and subsequent neuronal degeneration seen in Alzheimer's disease (AD). We previously demonstrated that the standardized Ginkgo biloba extract EGb 761, commonly used to enhance memory and by AD patients for dementia, inhibits Abeta-induced apoptosis in neuroblastoma cells. In this study, we use EGb 761 and its single constituents to associate Abeta species with Abeta-induced pathological behaviors in a model organism, Caenorhabditis elegans. We report that EGb 761 and one of its components, ginkgolide A, alleviates Abeta-induced pathological behaviors, including paralysis, and reduces chemotaxis behavior and 5-HT hypersensitivity in a transgenic C. elegans. We also show that EGb 761 inhibits Abeta oligomerization and Abeta deposits in the worms. Moreover, reducing oxidative stress is not the mechanism by which EGb 761 and ginkgolide A suppress Abeta-induced paralysis because the antioxidant L-ascorbic acid reduced intracellular levels of hydrogen peroxide to the same extent as EGb 761, but was not nearly as effective in suppressing paralysis in the transgenic C. elegans. These findings suggest that (1) EGb 761 suppresses Abeta-related pathological behaviors, (2) the protection against Abeta toxicity by EGb 761 is mediated primarily by modulating Abeta oligomeric species, and (3) ginkgolide A has therapeutic potential for prevention and treatment of AD.

Molecular dynamics simulations of the cytolytic toxin Cyt1A in solution.

Cytolytic toxin Cyt1A from Bacillus thuringiensis var. israelensis is used as an environmentally friendly insecticide, but its mode of action has not been clearly established. One main obstacle seems to be the lack of the experimentally determined structure of the toxin. As a first step in computer simulations of Cyt1A, in this paper, a three-dimensional molecular structure of Cyt1A in solution was generated by homology modeling, potential energy minimization and molecular dynamics. Regions of the toxin molecule that manifest increased conformational flexibility--and thus are likely to participate in the initial membrane binding and conformational changes--were then identified. Finally, the simulated structure was used to study the effect of a single amino-acid mutation that is known to abrogate the toxicity of Cyt1A in vivo.

A detergent-like mechanism of action of the cytolytic toxin Cyt1A from Bacillus thuringiensis var. israelensis.

The cytolytic delta-endotoxin Cyt1A from Bacillus thuringiensis var. israelensis is used in commercial preparations of environmentally safe insecticides. The current hypothesis on its mode of action is that the toxin self-assembles into well-defined cation-selective channels or pores, which results in colloid-osmotic lysis of the cell. Recently, a new hypothesis has been put forward suggesting that Cyt1A rather nonspecifically aggregates on the membrane surface and acts in a detergent-like manner. To distinguish between these two hypotheses, we investigated whether in the presence of lipid Cyt1A self-assembles into stoichiometric oligomers, which are characteristic of pores or channels, or aggregates into nonstoichiometric complexes, which would support the detergent-like model. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed that in the presence of lipid Cyt1A forms protein aggregates with a broad range of molecular weights, some being too large to enter the gel. Cyt1A tryptophan (Trp) fluorescence in the presence of lipid exhibited a decrease in anisotropy and quantum yield, but an unchanged lifetime, which is consistent with the presence of toxin aggregates in the membrane. Electrostatic interactions between the charged amino acid residues and the lipid headgroups are responsible for bringing the protein to the membrane surface, while hydrophobic and/or van der Waals interactions make the membrane binding irreversible. Fluorescence photobleaching recovery, a technique that measures the diffusion coefficient of fluorescently labeled particles, and epifluorescence microscopy revealed that upon addition of Cyt1A lipid vesicles were broken into smaller, faster diffusing objects. Since no change in size or morphology of the vesicles is expected when pores are formed in the osmotically equilibrated membranes, our results support the detergent-like mode of action of Cyt1A.

Effect of pH and ionic strength on the cytolytic toxin Cyt1A: a fluorescence spectroscopy study.

Cyt1A is a cytolytic toxin produced by Bacillus thuringiensis var. israelensis. Due to its toxicity in vivo against mosquitoes and black flies, it is used as an environmentally friendly insecticide, although its mode of action is not completely understood. The toxin is membrane-active, but its membrane-bound conformation is unknown. In the absence of direct structural data, fluorescence spectroscopy was used to obtain indirect information on Cyt1A conformation changes in the environment mimicking the vicinity of the lipid membrane (lower pH and increased ionic strength). With decreasing pH, Cyt1A's surface hydrophobicity increased, which is consistent with an increased interaction with model membranes at low pH values, as observed previously. The pK(a) value of this conformation change is 4.4+/-0.1. Intrinsic tryptophan fluorescence decreased with decreasing pH, and the pK(a) value was the same as the one determined with synthetic probes. The protein has two types of hydrophobic binding sites, and at low pH these sites bind more probe molecules (bis-ANS) with a higher affinity than at pH 7.4. When bound to the lipid, the toxin exhibited conformation similar to the molten-globule state and showed some characteristics also observed at low pH. However, the conformation of the lipid-bound toxin did not depend on pH. Neutral salts like NaCl and KCl induced conformational changes at neutral pH, but not at low pH. These changes were most probably due to specific interactions of the salt ions with the charged amino acids on the protein surface rather than due to general effects such as Hofmeister and Debye-Hückel. Our results might contribute to elucidating the mode of action of Cyt1A, and perhaps also to improving the formulation of the insecticidal preparations.

Experimental evidence for multiple assembled states of Sc3 from Schizophyllum commune.

The hydrophobin Sc3 from the fungus Schizophyllum commune assembles from the aqueous phase into ordered structures with substantially different characteristics depending upon experimental conditions. Under the first condition, a vortexing procedure widely reported in the literature, interfacial assembly yields highly ordered, stacked beta-sheets. We have also observed a previously unreported assembly of Sc3 under a second condition, which occurs in a time-dependent manner from quiescent solution. The resulting types of assembled states have been compared utilizing fluorescence techniques, sodium dodecyl sulfate polyacrylamide gel electrophoresis, immunoblotting, density gradient centrifugation, and phase contrast and atomic force microscopy. A model based on this study and previous literature is proposed that suggests three distinct states of Sc3: (1) soluble Sc3 consisting of unimers or multimers in micelle-like association, (2) interfacially assembled I-Sc3 with highly ordered, stacked beta-sheets, presumably formed in a templated manner at the air/water interface of microscopic bubbles generated by vortexing, and (3) solution-assembled S-Sc3, a less-ordered structure formed in a time-dependent manner in the absence of an interface.

Ginkgo biloba extract EGb 761 increases stress resistance and extends life span of Caenorhabditis elegans.

EGb 761, a standardized extract of Ginkgo biloba leaves, has been used in clinical trials for its beneficial effects on brain functions. In mammals, EGb 761 has been shown to enhance cognition, stress resistance, and longevity, but its molecular and cellular mechanisms are not known. In the present investigation, we used the model organism Caenorhabditis elegans to evaluate pharmacological effects of EGb 761 on aging. We tested the theory that EGb 761 augments the natural antioxidant system of C elegans, and thus increases stress resistance and longevity. We found that treatment of the wild-type worms with EGb 761 extended their median life span by 8%. Amongst several purified components of EGb 761, the flavonoid tamarixetin showed the most dramatic effect: it extended the median life span by 25%. Furthermore, EGb 761 increased the wild type's resistance to acute oxidative and thermal stress by 33% and 25%, respectively. Treatment of the prematurely aging mutant worms mev-1 with EGb 761 increased their resistance to acute oxidative and thermal stress by 33% and 11%, respectively. It appears that oxidative stress, a major determinant of life span, as well as other types of stress, can be successfully counteracted by the Ginlkgo biloba extract EGb 761.

Inhibition of amyloid-beta aggregation and caspase-3 activation by the Ginkgo biloba extract EGb761.

Standardized extract from the leaves of the Ginkgo biloba tree, labeled EGb761, has been used in clinical trials for its beneficial effects on brain functions, particularly in connection with age-related dementias and Alzheimer's disease (AD). Substantial experimental evidence indicates that EGb761 protects against neuronal damage from a variety of insults, but its cellular and molecular mechanisms remain unknown. Using a neuroblastoma cell line stably expressing an AD-associated double mutation, we report that EGb761 inhibits formation of amyloid-beta (Abeta) fibrils, which are the diagnostic, and possibly causative, feature of AD. The decreased Abeta fibrillogenesis in the presence of EGb761 was observed both in the conditioned medium of this Abeta-secreting cell line and in solution in vitro. In the cells, EGb761 significantly attenuated mitochondrion-initiated apoptosis and decreased the activity of caspase 3, a key enzyme in the apoptosis cell-signaling cascade. These results suggest that (i) neuronal damage in AD might be due to two factors: a direct Abeta toxicity and the apoptosis initiated by the mitochondria; and (ii) multiple cellular and molecular neuroprotective mechanisms, including attenuation of apoptosis and direct inhibition of Abeta aggregation, underlie the neuroprotective effects of EGb761.