The response of giant phospholipid vesicles to pore-forming peptide melittin

The interaction between the pore-forming peptide melittin (MLT) and giant phospholipid vesicles was explored experimentally. Micromanipulation and direct optical observation of a vesicle (loaded with sucrose solution and suspended in isomolar glucose solution) enabled the monitoring of a single vesicle response to MLT. Time dependences of the vesicle size, shape and the composition of the inner solution were examined at each applied concentration of MLT (in the range from 1 to 60 microg/ml). The response varied with MLT concentration from slight perturbation of the membrane to disintegration of the vesicle. A model for MLT-vesicle interaction is proposed that explains the observed phenomena in the entire span of MLT concentrations and is consistent with deduced underlying mechanisms of MLT action: trans-membrane positioning and dimerization of MLT, the lipid flow from the outer to the inner membrane leaflet induced by MLT translocation, formation of pores and the consequent transport of small molecules through the membrane. The results of the theoretical analysis stress the role of dimers in the MLT-membrane interaction and demonstrate that the MLT-induced membrane permeability for sugar molecules in this experimental set-up depends on both MLT concentration and time.     

Interlaboratory quality control in gynecologic cytopathology using the novel CONQUISTADOR software. Interobserver reproducibility in the Latin American screening study

OBJECTIVE: The recently developed software (CONQUISTADOR), capable of computing all intralaboratory and interlaboratory quality control (QC) indicators, was used to evaluate the diagnostic agreement among 4 cytology laboratories participating in the LAMS Study. STUDY DESIGN: The study was an interlaboratory exchange of specially designed 5 slide sets, each comprising 20 (conventional cytology) slides. At the first step, 80 slides (with "clear-cut" cases) were divided into four sets (A, B, C, D) of 20 specimens, each including inadequate and negative cases as well as in different proportions of all diagnostic TBS 2001 categories. In the second round, a fifth set (E) of 20 slides ("difficult cases") was designed, with all diagnostic categories, ASC and AGC included. Common measures of reproducibility (kappa and weighted kappa), accuracy (SE, SP, PPV, NPV) and 3 indices of diagnostic variability were calculated for sets A-D and set E, separately. RESULTS: For the 5 slide sets together, the weighted kappa was 0.8 (95% CI 0.76-0.85), which is the lower limit of the "almost perfect" ranking of kappa statistics, indicating an excellent interlaboratory agreement. The interlaboratory reproducibility was lower only for the difficult set (E). Similarly, the sensitivity for set E (70.0%) was lower than that (92.1%) for sets A-D. The diagnostic variability indices were not substantially different between the difficult (set E) and clearcut (sets A-D) cases. CONCLUSION: High interlaboratory reproducibility was obtained for sets A-D ("clear-cut" cases), while more interlaboratory variation was evident in the difficult samples. The new CONQUISTADOR software is a valuable tool in calculating the indicators needed in this intralaboratory and interlaboratory.     

Engineered staphylococcal protein A's IgG-binding domain with cathepsin L inhibitory activity

Inhibitory peptide of papain-like cysteine proteases, affinity selected from a random disulfide constrained phage-displayed peptide library, was grafted to staphylococcal protein A's B domain. Scaffold protein was additionally modified in order to allow solvent exposed display of peptide loop. Correct folding of fusion proteins was confirmed by CD-spectroscopy and by the ability to bind the Fc-region of rabbit IgG, a characteristic of parent domain. The recombinant constructs inhibited cathepsin L with inhibitory constants in the low-micromolar range.     

Use of Collagen Gel as a Three-Dimensional In Vitro Model to Study Electropermeabilization and Gene Electrotransfer

Gene electrotransfer is a promising nonviral method that enables transfer of plasmid DNA into cells with electric pulses. Although many in vitro and in vivo studies have been performed, the question of the implied gene electrotransfer mechanisms is largely open. The main obstacle toward efficient gene electrotransfer in vivo is relatively poor mobility of DNA in tissues. Since cells are mechanically coupled to their extracellular environment and act differently compared to standard in vitro conditions, we developed a three-dimensional (3-D) in vitro model of CHO cells embedded in collagen gel as an ex vivo model of tissue to study electropermeabilization and different parameters of gene electrotransfer. For this purpose, we first used propidium iodide to detect electropermeabilization of CHO cells embedded in collagen gel. Then, we analyzed the influence of different concentrations of plasmid DNA and pulse duration on gene electrotransfer efficiency. Our results revealed that even if cells in collagen gel can be efficiently electropermeabilized, gene expression is significantly lower. Gene electrotransfer efficiency in our 3-D in vitro model had similar dependence on concentration of plasmid DNA and pulse duration comparable to in vivo studies, where longer (millisecond) pulses were shown to be more optimal compared to shorter (microsecond) pulses. The presented results demonstrate that our 3-D in vitro model resembles the in vivo situation more closely than conventional 2-D cell cultures and, thus, provides an environment closer to in vivo conditions to study mechanisms of gene electrotransfer.     

Surface plasmon resonance in protein-membrane interactions

Surface plasmon resonance (SPR) has become one of the most important techniques for studying macromolecular interactions. The most obvious advantages of SPR over other techniques are: direct and rapid determination of association and dissociation rates of binding process, no need for labelling of protein or lipids, and small amounts of sample used in the assay (often nM concentrations of proteins). In biochemistry, SPR is used mainly to study protein-protein interactions. On the other hand, protein-membrane interactions, although crucial for many cell processes, are less well studied. Recent advances in the preparation of stable membrane-like surfaces and the commercialisation of sensor chips has enabled widespread use of SPR in protein-membrane interactions. One of the most popular is Biacore's L1 sensor chip that allows capture of intact liposomes or even subcellular preparations. Lipid specificity of protein-membrane interactions can, therefore, be easily studied by manipulating the lipid composition of the immobilised membrane. The number of published papers has increased steadily in the last few years and the examples include domains or proteins that participate in cell signalling, pore-forming proteins, membrane-interacting peptides, coagulation factors, enzymes, amyloidogenic proteins, prions, etc. This paper gives a brief overview of different membrane-mimetic surfaces that can be prepared on the surface of SPR chips, properties of liposomes on the surface of L1 chips and some selected examples of protein-membrane interactions studied with such system.     

Chemoporation using saponins or cholates: an alternative method for transformation of bacterial cells

A new method for fast transformation of competent bacterial cells has been developed. The transformation is induced with cholic acid analogues or saponins which cause reversible disruption of the bacterial membrane. This method shortens the time of transformation without significant loss of transformation efficiency in comparison to heat shock method and is the first reported chemically-induced transformation. New data about interactions between cholates and biomembranes is revealed that may contribute to better understanding of bacterial transformation.     

Electro‐mediated gene transfer and expression are controlled by the life‐time of DNA/membrane complex formation

Background

Electroporation is a physical method used to transfer molecules into cells and tissues. Clinical applications have been developed for antitumor drug delivery. Clinical trials of gene electrotransfer are under investigation. However, knowledge about how DNA enters cells is not complete. By contrast to small molecules that have direct access to the cytoplasm, DNA forms a long lived complex with the plasma membrane and is transferred into the cytoplasm with a considerable delay.

Methods

To increase our understanding of the key step of DNA/membrane complex formation, we investigated the dependence of DNA/membrane interaction and gene expression on electric pulse polarity and repetition frequency.

Results

We observed that both are affected by reversing the polarity and by increasing the repetition frequency of pulses. The results obtained in the present study reveal the existence of two classes of DNA/membrane interaction: (i) a metastable DNA/membrane complex from which DNA can leave and return to external medium and (ii) a stable DNA/membrane complex, where DNA cannot be removed, even by applying electric pulses of reversed polarity. Only DNA belonging to the second class leads to effective gene expression.

Conclusions

The life‐time of DNA/membrane complex formation is of the order of 1 s and has to be taken into account to improve protocols of electro‐mediated gene delivery. Copyright © 2009 John Wiley & Sons, Ltd.     

Changing the Direction and Orientation of Electric Field During Electric Pulses Application Improves Plasmid Gene Transfer in vitro

Gene electrotransfer is a physical method used to deliver genes into the cells by application of short and intense electric pulses, which cause destabilization of cell membrane, making it permeable to small molecules and allows transfer of large molecules such as DNA. It represents an alternative to viral vectors, due to its safety, efficacy and ease of application. For gene electrotransfer different electric pulse protocols are used in order to achieve maximum gene transfection, one of them is changing the electric field direction and orientation during the pulse delivery. Changing electric field direction and orientation increase the membrane area competent for DNA entry into the cell. In this video, we demonstrate the difference in gene electrotransfer efficacy when all pulses are delivered in the same direction and when pulses are delivered by changing alternatively the electric field direction and orientation. For this purpose tip with integrated electrodes and high-voltage prototype generator, which allows changing of electric field in different directions during electric pulse application, were used. Gene electrotransfer efficacy is determined 24h after pulse application as the number of cells expressing green fluorescent protein divided with the number of all cells. The results show that gene transfection is increased when the electric field orientation during electric pulse delivery is changed.Download video file.^{(27M, mov)}     

Inhibition of AChE by malathion and some structurally similar compounds

Inhibition of bovine erythrocyte acetylcholinesterase (free and immobilized on controlled pore glass) by separate and simultaneous exposure to malathion and malathion transformation products which are generally formed during storage or through natural or photochemical degradation was investigated. Increasing concentrations of malathion, its oxidation product malaoxon, and its isomerisation product isomalathion inhibited free and immobilized AChE in a concentration-dependent manner. K_I, the dissociation constant for the initial reversible enzyme inhibitor-complex, and k₃, the first order rate constant for the conversion of the reversible complex into the irreversibly inhibited enzyme, were determined from the progressive development of inhibition produced by reaction of native AChE with malathion, malaoxon and isomalathion. K_I values of 1.3 × 10^{? 4} M^{? 1}, 5.6 × 10^{? 6} M^{? 1} and 7.2 × 10^{? 6} M^{? 1} were obtained for malathion, malaoxon and isomalathion, respectively. The IC₅₀ values for free/immobilized AChE, (3.7 ± 0.2) × 10^{? 4} M/(1.6 ± 0.1) × 10^{? 4}, (2.4 ± 0.3) × 10^{? 6}/(3.4 ± 0.1) × 10^{? 6} M and (3.2 ± 0.3) × 10^{? 6} M/(2.7 ± 0.2) × 10^{? 6} M, were obtained from the inhibition curves induced by malathion, malaoxon and isomalathion, respectively. However, the products formed due to photoinduced degradation, phosphorodithioic O,O,S-trimethyl ester and O,O-dimethyl thiophosphate, did not noticeably affect enzymatic activity, while diethyl maleate inhibited AChE activity at concentrations > 10 mM. Inhibition of acetylcholinesterase increased with the time of exposure to malathion and its inhibiting by-products within the interval from 0 to 5 minutes. Through simultaneous exposure of the enzyme to malaoxon and isomalathion, an additive effect was achieved for lower concentrations of the inhibitors (in the presence of malaoxon/isomalathion at concentrations 2 × 10^{? 7} M/2 × 10^{? 7} M, 2 × 10^{? 7} M/3 × 10^{? 7} M and 2 × 10^{? 7} M/4.5 × 10^{? 7} M), while an antagonistic effect was obtained for all higher concentrations of inhibitors. The presence of a non-inhibitory degradation product (phosphorodithioic O,O,S-trimethyl ester) did not affect the inhibition efficiencies of the malathion by-products, malaoxon and isomalathion.