Vesicle fusion with bilayer lipid membrane controlled by electrostatic interaction

The fusion of proteoliposomes is a promising approach for incorporating membrane proteins in artificial lipid membranes. In this study, we employed an electrostatic interaction between vesicles and supported bilayer lipid membranes (s-BLMs) to control the fusion process. We combined large unilamellar vesicles (LUVs) containing anionic lipids, which we used instead of proteoliposomes, and s-BLMs containing cationic lipids to control electrostatic interaction. Anionic LUVs were never adsorbed or ruptured on the SiO₂ substrate with a slight negative charge, and selectively fused with cationic s-BLMs. The LUVs can be fused effectively to the target position. Furthermore, as the vesicle fusion proceeds and some of the positive charges are neutralized, the attractive interaction weakens and finally the vesicle fusion saturates. In other words, we can control the number of LUVs fused with s-BLMs by controlling the concentration of the cationic lipids in the s-BLMs. The fluidity of the s-BLMs after vesicle fusion was confirmed to be sufficiently high. This indicates that the LUVs attached to the s-BLMs were almost completely fused, and there were few intermediate state vesicles in the fusion process. We could control the position and amount of vesicle fusion with the s-BLMs by employing an electrostatic interaction.     

类脂双层(BLM)概念及其实验进展——BLM的40年

类脂双层研究的动力是来自生物世界.虽然1961年首次报道生物体外的双层类脂膜（BLMs）自组装,但从Hooke (1672年)起,实验科学家一直在处理有关BLM类型的界面吸附现象.BLMs（或平面的类脂双层）已应用在许多方面,即从基础的膜生物物理学到实际的爱滋病研究,从通过水光分解的太阳能转换,到应用支持双层类脂膜(s-BLMs)的生物传感器的发展,到包括细胞凋亡（apoptosis）在内的光生物学.综述了类脂双层概念的起源及其实验进展,以及BLMs用作某些生物膜模型的最新研究.此外,简要描述近来有关通过s-BLMs来发展生物传感器的工作.     

A minisensor for the rapid screening of sucralose based on surface-stabilized bilayer lipid membranes.

This work describes an electrochemical technique that is suitable for the rapid and sensitive screening of the sweetener sucralose based on surface-stabilized bilayer lipid membranes (s-BLMs) composed of egg phosphatidylcholine. The interactions of sucralose with s-BLMs produced electrochemical ion current increases, which appeared reproducible within a few seconds after exposure of the membranes to the sweetener. The mechanism of signal generation was investigated by differential scanning calorimetric studies. The mechanism was found to be associated with alteration of the electrostatic fields of the lipid film. These studies revealed that an increase of the molecular area of the lipids at the membranes and a stabilization of a gel phase structure occurred due to adsorption of the sweetener. Water molecules are adsorbed at the polar headgroups of the lipids, which changes the electrostatic field at the surface of the membranes. The current signal increases were related to the concentration of sucralose in bulk solution in the micromolar range. The present lipid film based sensor provided a fast response (i.e. in the order of a few seconds) to alterations of sucralose concentration (5-50 microm) in electrolyte solution. The electrochemical transduction of the interactions of this artificial sweetener with s-BLMs was applied in the determination of this compound in granulated sugar substitute products using the present minisensor.     

Self-assembling bilayer lipid membranes on solid support

Solid-supported bilayer lipid membranes (s-BLMs) that possess some properties similar to those of conventional BLMs can be self-assembled on a freshly cleaved metal wire by a two-step procedure: (i) The tip of a Teflon-coated platinum wire, while immersed in a lipid solution, is cut off with a scalpel; (ii) the new tip of the wire, having become coated with lipid solution, is transferred into 0.1 M KCl. After a few minutes, a stable lipid bilayer forms spontaneously on the tip of the wire, as verified by electrical measurements. An application of such a supported BLM (s-BLM) is reported for the detection of Pb2+ ions. The s-BLM is liquid-crystalline in structure, which makes it amenable to modification for basic studies, as well as for technological applications such as biosensors and molecular electronic devices.     

Conducting polymer polypyrrole supported bilayer lipid membranes

Electrochemically synthesized conducting polymer polypyrrole (PPy) film on gold electrode surface was used as a novel support for bilayer lipid membranes (BLMs). Investigations by surface plasmon resonance (SPR) suggest that dimyristoyl-L-alpha-phosphatidylcholine (DMPC) and dimyristoyl-L-alpha-phosphatidyl-L-serine (DMPS) can form BLMs on PPy film surface but dimyristoyl-L-alpha-phosphatidylglycerol (DMPG) and didodecyldimethylammonium bromide (DDAB) can not do so, indicating the formation of PPy supported bilayer lipid membranes (s-BLMs) is dependent on the chemical structure of the lipids used. The self-assembly of DMPC induces a smoother topography than the PPy layer with rms roughness decreasing from 4.484 to 2.914 nm convinced by atomic force microscopy (AFM). Impedance spectroscopy measurements confirm that the deposition of BLM substantially increases the resistance of the system indicating a very densely packed BLM structures. The little change of PPy film in capacitance shows that solvent and electrolyte ions still retain within the porous PPy film after BLM deposition. Therefore, the PPy supported BLM is to some extent comparable to conventional BLM with aqueous medium retaining at its two sides. As an example and preliminary application, horseradish peroxidase (HRP) reconstituted into the s-BLM shows the expected protein activity and can transfer electron from or to the underlying PPy support for its response to electrocatalytic reduction of hydrogen peroxide in solution. Thus the system maybe possesses potential applications to biomimetic membrane studies.     

Fullerene modified supported lipid membrane as sensitive element of sensor for odorants

A supported lipid bilayer membrane (s-BLMs) formed on a freshly cleaved metallic surface by the Tien method was applied for the design of an electrochemical sensor for detection of neutral odorant molecules. The lipid bilayer was modified by saturation with fullerene C₆₀, which possesses electron mediator properties and facilitates a redox reaction occurring at the border of the lipid membrane and metal surface. I₂/I⁻ and ferrocenyl trimethyl bromide were used as electroactive marker ions. The smell compounds adsorb on the surface of the lipid layer and change its structure. As a consequence the ratio of marker ion penetration to the lipid membrane is altered. The magnitude of these changes depends on the amount and chemical structure of adsorbed molecules. The research presented was carried out by cyclic voltammetry. The magnitude of the electrochemical signal generated by smell compounds was correlated with other parameters describing their molecular properties such as: octanol/water partition coefficients and dipole moments.     

Significant proteins affecting cerebral vasospasm using complementary ICPMS and MALDI-MS

Cerebral vasospasm (CV) following subarachnoid hemorrhagic stroke affects more than one million people each year. The etiology and prevention of CV is currently of great interest to researchers in various fields of medical science. More recently, the idea that selenium could be playing a major role in the onset of cerebral vasospasm has come into the spotlight. This study focused on using newly established metallomics techniques in order to explore the proteome associated with CV and if selenium might affect the discovered proteins. Size exclusion chromatography coupled to inductively coupled plasma mass spectrometry, along with LC-MALDI-TOF/TOF were both essential in determining protein identifications in three different sample types; a control (normal, healthy patient, CSF control), SAH stroke patients (no vasospasm, CSF C) and SAH CV patients (CSF V). The results of this study, although preliminary, indicate the current methods are applicable and warrant further application to these clinically important targets.     

Impedance spectroscopy of bilayer lipid membranes self-assembled on agar support - interaction with HDL

The electrical properties of the supported lipid bilayer membrane (s-BLM) of egg phosphatidylcholine (PC) self-assembled on agar surface were examined. To characterize the insulating properties of s-BLMs, electrochemical impedance spectroscopy (EIS) was used. The analysis of impedance spectra in terms of an equivalent circuit of agar/electrolyte and agar/s-BLM/electrolyte in the frequency range of 0.1 Hz-10 kHz was performed. The high-density lipoproteins (HDL)/s-BLM interaction in the concentration range from 20 microg/ml to 80 microg/ml of HDL was investigated. It is evident that treatment of s-BLM with HDL resulted in an increase of the lipid film resistance and a decrease of membrane capacitance.     

The effect of temperature on conduction velocity in human muscle fibers

The effects of variation of intramuscular temperature (T) on conduction velocity (CV) of the action potential along single human muscle fibers of the biceps brachii was studied in situ in 15 normal volunteers (mean age 39 years, range 21–62 years). Cooling was obtained by direct application of ice over a rectangular skin region including the stimulating and recording area. The intramuscular T was monitored by a needle thermocouple (copperconstantane). In all the 24 muscle fibers studied, a linear relationship was observed between CV and T. The slopes of the regression lines, ranging between 0.190 and 0.079 m/s, were positively correlated with the starting CV at 36°C ranging between 2.2 and 5.2 m/s. If conduction changes are expressed as a percentage of the basal CV at 36°C, the CV/T coefficient is the same for all the fibers and independent of the individual CV: 3.4% of CV/°C.     

The relationship between mean channel selection and the calculated coefficient of variation

Calculated coefficients of variation (CV) taken from the quotient of the standard deviation (S.D.) and the mean value of measured distributions are often used as an indicator of system performance in linear flow cytometry (FCM). The ability of the calculated CV to estimate the true CV of the underlying experiment before grouping (channelization) is dependent on the relationship between the width of the data channels and the magnitude of the S.D. of the measured distribution. When the channel width is equal to the S.D. of a distribution, the calculated CV is approximately 20% larger than the true CV of an experiment. By the time the S.D. is only one-half of a channel width, the calculated CV is unreliable. When the distribution S.D. is narrower than a channel's width, small changes in the distribution mean value will cause large variations in the calculated CV. As the true CV decreases, the calculation must be made with higher mean channel values. This dependence of calculated CV accuracy upon the relationship between S.D. and channel width places limitations upon mean channel selection that must be considered when using CV calculations for evaluating system performance, especially when looking for small improvements during optical alignment procedures. When an instrument is assumed to have a constant CV and the data are collected linearly, it is possible to improve the CV estimation accuracy by placing distributions in higher-numbered channels.(ABSTRACT TRUNCATED AT 250 WORDS)     

The effects of bipolar electrode montage on conduction velocity estimation from the surface electromyogram.

This study examines the influence of the bipolar electrode montage on conduction velocity (CV) estimation. Electrode montage refers to the combination of two parameters, the inter-electrode distance (IED), the distance between the two electrodes of a bipolar pair, and the inter-signal distance (ISD), the distance between two bipolar signals used to calculate CV. Data from the biceps brachii (BB) and tibialis anterior (TA) of healthy subjects are analysed. Two approaches are used for CV estimation. The first returns a single value per epoch. The second is based on finding velocity values from individual peaks in the signal and results in a peak velocity (PV) distribution being generated per epoch. It is concluded that CV estimation is significantly dependent on the choice of the (IED, ISD) electrode montage. The main results are that the electrode montage affects (1) the mean PV and CV estimates, (typically P < 0.001), (2) the degree of spatial variability, and (3) the width of the PV distributions. The combination of a small IED and large an ISD is recommended.     

Hormonal effects of CV 205-502, a novel octahydrobenzo [g] quinoline with potent dopamine agonist properties

Following the success of ergot alkaloids and their synthetic derivatives in treating a variety of pathophysiological disturbances, efforts have been concentrated on the synthesis of new derivatives and partial structures with the aim of dissecting out a specifically dopaminomimetic pharmacophore. Accordingly CV 205-502, a structure which superposes the linear benzo [g] quinoline segment of apomorphine on the substituted pyrrolo [3,4- g] quinoline moiety of the ergolines was designed. This study was performed in normal young volunteers to investigate the effect of single oral doses of CV 205-502 on plasma prolactin levels and on other endocrine parameters (GH, LH, FSH, TSH and cortisol) as well as on tolerability. 10 volunteers participated in a dose-ranging study. They received single oral doses of 0.01 to 0.09 mg CV 205-502, in order to assess the prolactin suppressant action. 6 volunteers were given a dose of 0.06 mg CV 205-502 in order to determine the endocrine profile of the compound. The duration of action of 0.06 mg CV 205-502 was investigated in 6 subjects by measuring plasma PRL and GH levels for 48 h. The results show strong dose-dependent suppression of PRL appearing following doses between 0.04 and 0.09 mg of CV 205-502. PRL was markedly suppressed for more than 24h. and the peaks of the normal sleep profile were abolished. Intermittent transient GH increases occurred during the first 6 hours; sleep profiles were normal. Compared with placebo values, no changes were seen in the levels of any other hormone except TSH, which decreased. Tolerability was good and no drug attributable changes in safety measures occurred. This study demonstrates that CV 205-502 is a potent and long acting PRL suppressant compound and suggest that this novel octahydrobenzo [g] quinoline will prove to be a therapeutically useful dopaminomimetic compound.     

The contractile vacuole and its membrane dynamics

The contractile vacuole (CV) is an osmoregulatory organelle whose mechanisms of function are poorly understood. Immunological studies in the last decade have demonstrated abundant proton-translocating V-type ATPases (V-ATPases) in its membrane that could provide the energy, from proton electrochemical gradients, for moving ions into the CV to be followed by water. This review emphasizes recent work on the contractile vacuole complex (CVC) of Paramecium including (1) CV expulsion, (2) a role for V-ATPases in sequestering fluid, (3) identifying ions in the cytosol and in the CV, (4) in situ electrophysiological parameters of the CVC membrane, and (5) a better understanding of the membrane dynamics of this organelle.     

Determination of immunoreactivity of doxorubicin antibody immunoconjugates by a Le(y) competitive RIA.

Many monoclonal antibody-drug immunoconjugates have been evaluated for their ability to deliver cytotoxic drugs to tumors. It is essential to establish that the ability of the conjugates to bind antigen, i.e. their immunoreactivity, is not adversely affected by the drug conjugation procedure. We have described herein a measurement of the immunoreactivity of BR96-DOX, a conjugate comprised of BR96, a chimeric monoclonal antibody specific for the Le(y) tetrasaccharide commonly expressed on human carcinomas, and doxorubicin, an anticancer agent in widespread clinical use. We have employed a competitive RIA, in which microtiter wells were coated with synthetic Le(y) conjugated to human serum albumin and then incubated with 125I-labeled antibody BR96 in the presence of test conjugate or intact BR96 mAb. The test conjugates were found to compete as effectively as unconjugated BR96. This assay is highly applicable to QC processes with the intra-assay CV = 2.0% and the interassay CV = 4.3%.     

Surface EMG recorded by branched electrodes during sustained muscle activity.

The purpose of the present investigation is to use surface interference EMG recorded by branched electrodes for assessment of muscle fatigue during sustained voluntary isometric contractions at different levels. Level-trigger averaging and turn/amplitude analysis have been applied. The conduction velocity (CV) of excitation was calculated from the time shift of the negative peaks of the averaged potentials (AvPs) derived from the EMG recorded by two electrodes placed along the muscle fibers. The recruitment of new motor units affects the negative amplitude (NA) of AvPs, the number of turns per second and the mean amplitude of turns in a different way depending on the level of sustained contractions. In contrast, the CV declined at all levels of sustained contractions and was the most appropriate parameter for the muscle fatigue assessment. There was a good correlation between CV decrease and torque reduction during sustained maximal efforts. The level-trigger averaging technique of the interference EMG recorded by surface branched electrodes is easy and non-invasive, thus being very convenient for routine application.     

Precision of nanoindentation protocols for measurement of viscoelasticity in cortical and trabecular bone

Nanoindentation has recently gained attention as a characterization technique for mechanical properties of biological tissues, such as bone, on the sub-micron level. However, optimal methods to characterize viscoelastic properties of bones are yet to be established. This study aimed to compare the time-dependent viscoelastic properties of bone tissue obtained with different nanoindentation methods. Bovine cortical and trabecular bone samples (n=8) from the distal femur and proximal tibia were dehydrated, embedded and polished. The material properties determined using nanoindentation were hardness and reduced modulus, as well as time-dependent parameters based on creep, loading-rate, dissipated energy and semi-dynamic testing under load control. Each loading protocol was repeated 160 times and the reproducibility was assessed based on the coefficient of variation (CV). Additionally, three well-characterized polymers were tested and CV values were calculated for reference.The employed methods were able to characterize time-dependent viscoelastic properties of bone. However, their reproducibility varied highly (CV 9–40%). The creep constant increased with increasing dwell time. The reproducibility was best with a 30 s creep period (CV 18%). The dissipated energy was stable after three repeated load cycles, and the reproducibility improved with each cycle (CV 23%). The viscoelastic properties determined with semi-dynamic test increased with increase in frequency. These measurements were most reproducible at high frequencies (CV 9–10%). Our results indicate that several methods are feasible for the determination of viscoelastic properties of bone material. The high frequency semi-dynamic test showed the highest precision within the tested nanoindentation protocols.     

Voltage Biasing, Cyclic Voltammetry, & Electrical Impedance Spectroscopy for Neural Interfaces

Electrical impedance spectroscopy (EIS) and cyclic voltammetry (CV) measure properties of the electrode-tissue interface without additional invasive procedures, and can be used to monitor electrode performance over the long term. EIS measures electrical impedance at multiple frequencies, and increases in impedance indicate increased glial scar formation around the device, while cyclic voltammetry measures the charge carrying capacity of the electrode, and indicates how charge is transferred at different voltage levels. As implanted electrodes age, EIS and CV data change, and electrode sites that previously recorded spiking neurons often exhibit significantly lower efficacy for neural recording. The application of a brief voltage pulse to implanted electrode arrays, known as rejuvenation, can bring back spiking activity on otherwise silent electrode sites for a period of time. Rejuvenation alters EIS and CV, and can be monitored by these complementary methods. Typically, EIS is measured daily as an indication of the tissue response at the electrode site. If spikes are absent in a channel that previously had spikes, then CV is used to determine the charge carrying capacity of the electrode site, and rejuvenation can be applied to improve the interface efficacy. CV and EIS are then repeated to check the changes at the electrode-tissue interface, and neural recordings are collected. The overall goal of rejuvenation is to extend the functional lifetime of implanted arrays.     

GLP-1 receptor agonists and reduction of cardiometabolic risk: Potential underlying mechanisms

Type 2 diabetes mellitus (T2DM) is a metabolic condition with an elevated impact on cardiovascular (CV) risk. The innovative therapeutic approaches for T2DM - incretin-based therapies (IBTs), including glucagon-like peptide 1 (GLP-1) receptor agonists, have become popular and more widely used in recent years. The available scientific data from clinical studies and clinical practice highlights their beyond glucose-lowering effects, which is achieved without any increase in hypoglycaemia. The former effects include reduction in body weight, lipids, blood pressure, inflammatory markers, oxidative stress, endothelial dysfunction, and subclinical atherosclerosis, thus reducing and potentially preventing CV events. In fact, the introduction of IBTs is one of the key moments in the history of diabetes research and treatment. Such therapeutic strategies allow customization of antidiabetic treatment to each patient's need and therefore obtain better metabolic control with reduced CV risk. The aim of the present paper is to provide a comprehensive overview of the effects of GLP-1RA on various cardiometabolic markers and overall CV risk, with particular attention on recent CV outcome studies and potential mechanisms. In particular, the effects of liraglutide on formation and progression of atherosclerotic plaque and mechanisms explaining its cardioprotective effects are highlighted.     

<Emphasis Type="BoldItalic">Dictyostelium discoideum</Emphasis> RabS and Rab2 colocalize with the Golgi and contractile vacuole system and regulate osmoregulation

Small-molecular-weight GTPase Rab2 has been shown to be a resident of pre-Golgi intermediates and is required for protein transport from the ER to the Golgi complex; however, Rab2 has yet to be characterized in Dictyostelium discoideum. DdRabS is a Dictyostelium Rab that is 80% homologous to DdRab1 which is required for protein transport between the ER and Golgi. Expression of GFP-tagged DdRab2 and DdRabS proteins showed localization to Golgi membranes and to the contractile vacuole system (CV) in Dictyostelium. Microscopic imaging indicates that the DdRab2 and DdRabS proteins localize at, and are essential for, the proper structure of Golgi membranes and the CV system. Dominant negative (DN) forms show fractionation of Golgi membranes, supporting their role in the structure and function of it. DdRab2 and DdRabS proteins, and their dominant negative and constitutively active (CA) forms, affect osmoregulation of the cells, possibly by the influx and discharge of fluids, which suggests a role in the function of the CV system. This is the first evidence of GTPases being localized to both Golgi membranes and the CV system in Dictyostelium.     

Multi-vector composition and its application in landscape patch shape deformation and dynamic analysis

The quantitative expression and monitoring of patch shape deformation is an important content of landscape ecology. In this paper, a new multi-vector method is put forward based on curve representation in differential geometry, which is the composition of Centroid Displacement Vector (CDV), Local Deformation Vector (LDV) and the unit normal vector on the patch boundary. These composition vectors (CVs) not only add a new direction dimension to the landscape pattern metrics already available, but also provide multi-scale information, i.e. CV at every point and the boundary segment CV (BSCV). This new information can provide a quantification basis for a deeper understanding of the mechanisms of shape deformation and dynamic analysis. An application of CVs to patch dynamics in the Yancheng National Nature Reserve (YNNR) in China is given to validate the methodology. The susceptibility to invasion of the Common Seepweed in the core area of YNNR is evaluated quantitatively by CVs method. Our results indicate that the rate of invasion of Smooth Cord-grass into Common Seepweed patch has increased by 1.5 times from 1995 to 2002. This is considered to be the dominant factor causing Common Seepweed patches to shrink and change dynamically. These results help us better understand the landscape patch shape deformation and analyze the driver forces of landscape change, and they also provide the quantitative basis for the effective protection of the nature reserve area.