Hans-Joachim Galla

Piezoelectric Mass‐Sensing Devices as Biosensors—An Alternative to Optical Biosensors?

In the early days of electronic communication-as a result of the limited number of quartz resonators available-frequency adjustment was accomplished by a pencil mark depositing a foreign mass layer on the crystal. In 1959, Sauerbrey showed that the shift in resonance frequency of thickness-shear-mode resonators is proportional to the deposited mass. This was the starting point for the development of a new generation of piezoelectric mass-sensitive devices. However, it was the development of new powerful oscillator circuits that were capable of operating thickness shear mode resonators in fluids that enabled this technique to be introduced into bioanalytic applications. In the last decade adsorption of biomolecules on functionalized surfaces turned in to one of the paramount applications of piezoelectric transducers. These applications include the study of the interaction of DNA and RNA with complementary strands, specific recognition of protein ligands by immobilized receptors, the detection of virus capsids, bacteria, mammalian cells, and last but not least the development of complete immunosensors. Piezoelectric transducers allow a label-free detection of molecules; they are more than mere mass sensors since the sensor response is also influenced by interfacial phenomena, viscoelastic properties of the adhered biomaterial, surface charges of adsorbed molecules, and surface roughness. These new insights have recently been used to investigate the adhesion of cells, liposomes, and proteins onto surfaces, thus allowing the determination of the morphological changes of cells as a response to pharmacological substances and changes in the water content of biopolymers without employing labor-intense techniques. However, the future will show whether the quartz-crystal microbalance will assert itself against established label-free sensor devices such as surface plasmon resonance spectroscopy and interferometry.

NADPH Oxidase Plays a Central Role in Blood-Brain Barrier Damage in Experimental Stroke

Background and Purpose— Cerebral ischemia/reperfusion is associated with reactive oxygen species (ROS) generation, and NADPH oxidases are important sources of ROS. We hypothesized that NADPH oxidases mediate blood-brain barrier (BBB) disruption and contribute to tissue damage in ischemia/reperfusion. Methods— Ischemia was induced by filament occlusion of the middle cerebral artery in mice for 2 hours followed by reperfusion. BBB permeability was measured by Evans blue extravasation. Monolayer permeability was determined from transendothelial electrical resistance of cultured porcine brain capillary endothelial cells. Results— BBB permeability was increased in the ischemic hemisphere 1 hour after reperfusion. In NADPH oxidase–knockout (gp91phox−/−) mice, middle cerebral artery occlusion–induced BBB disruption and lesion volume were largely attenuated compared with those in wild-type mice. Inhibition of NADPH oxidase by apocynin prevented BBB damage. In porcine brain capillary endothelial cells, hypoxia/reoxygenation induced translocation of the NADPH oxidase activator Rac-1 to the membrane. In vivo inhibition of Rac-1 by the 3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitor atorvastatin or Clostridium difficile lethal toxin B also prevented the ischemia/reperfusion–induced BBB disruption. Stimulation of porcine brain capillary endothelial cells with H2O2 increased permeability, an effect attenuated by inhibition of phosphatidyl inositol 3-kinase or c-Jun N-terminal kinase but not blockade of extracellular signal–regulated kinase-1/2 or p38 mitogen-activated protein kinase. Inhibition of Rho kinase completely prevented the ROS-induced increase in permeability and the ROS-induced polymerization of the actin cytoskeleton. Conclusions— Activation of Rac and subsequently of the gp91phox containing NADPH oxidase promotes cerebral ROS formation, which then leads to Rho kinase–mediated endothelial cell contraction and disruption of the BBB. Inhibition of NAPDH oxidase is a promising approach to reduce brain injury after stroke.

On two-dimensional passive random walk in lipid bilayers and fluid pathways in biomembranes

SummaryThe lateral mobility of pyrene, pyrene decanoic acid, and 1-palmitoyl-2-pyrene decanoyl-phosphatidyl choline (pyrene lecithin) in lipid bilayers is determined by the excimer formation technique. This method is applied to vesicles of lecithins differing in chain length and in the degree of saturation of the hydrocarbon chains. These values are compared with results in cephalins of different chain length and in dipalmitoyl phosphatidic acid at variable pH. The influence of cholesterol is investigated. The results are analyzed in terms of the Montroll model of two-dimensional random walk. The jump frequency of the probe molecule within the lipid lattice is obtained. The advantage of this measure of transport in lipid layers is that it does not involve lipid lattice parameters.The main results of the present work are: (i) The lateral mobility of a given solute molecule in lamellae of saturated lecithins is independent of hydrocarbon chain length and rather a universal function of temperature. (ii) In unsaturated dioleyl lecithin the amphiphatic molecules have lateral mobilities of the same size as in saturated lipids. The jump frequency of pyrene, however, is by a factor of two larger in the unsaturated lecithin. (iii) The jump frequencies in phosphatidyl ethanolamines are about equal to those in lecithins. (iv) In phosphatidic acid layers the hopping frequencies depend on the chargers of the head groups of both the lipids and the probes. (v) Cholesterol strongly reduces the jump frequency in fluid layers. (vi) The lateral mobility in biological membranes is comparable to that in artificial lipid bilayers.The experimental results are discussed in terms of the free volume model of diffusion in fluids. Good agreement with the predictions made from this model is found. A striking result is the observation of a tilt in dioleyl-lecithin bilayer membranes from the hopping frequencies of pyrene and pyrene lecithin. A tilt angle of ϕ-17° is estimated.

Impedance analysis of supported lipid bilayer membranes: a scrutiny of different preparation techniques

One topic of this study is the comparison of different preparation techniques to build up solid supported lipid bilayers onto gold substrates. The deposited lipid bilayers were investigated by a.c. impedance spectroscopy. Three different strategies were applied: (1) The gold surface was initially covered with a chemisorbed monolayer of octadecanethiol or 1,2-dimyristoyl-sn-glycero-3-phosphothioethanol (DMPTE). The second monolayer consisting of phospholipids was then deposited onto this hydrophobic surface by (i) the Langmuir-Schaefer-technique, (ii) from lipid solution in n-decane/isobutanol, (iii) by the lipid/detergent dilution technique or (iv) by fusion of vesicles. (2) Charged molecules carrying thiol-anchors for attachment to the gold surface by chemisorption were used. Negatively charged surfaces of 3-mercaptopropionic acid were found to be excellent substrates that allow the attachment of planar lipid bilayers by applying positively charged dimethyldioctadecylammoniumbromide (DODAB) vesicles or negatively charged 1,2-dipalmitoyl-sn-glycero-3-phosphoglycerol vesicles in the presence of chelating Ca2+-ions. If positively charged first monolayers of mercaptoethylammoniumhydrochloride were used we were able to attach mixed 1,2-dimyristoyl-sn-glycero-3-phosphoglycerol/1,2-dimyristoyl-sn-glycero- 3-phosphoethanolamine vesicles to form planar lipid bilayers via electrostatic interaction. (3) Direct deposition of lipid bilayers is possible from vesicles containing 1,2-dimyristoyl-sn-glycero-3-phosphothioethanol (DMPTE). A critical amount of more than 50 mol% of DMPTE was found to be necessary to form a solid supported lipid bilayer. Bilayers obtained with these different preparation techniques were scrutinized with respect to their capacitances, kinetics of formation and their long-term stabilities by impedance spectroscopy. The second feature of this paper is the application of the supported bilayers to study ion transport through channel-forming peptides. We used a DODAB-bilayer for the reconstitution of gramicidin D channels. By circular dichroism measurements we verified that the peptide is in its channel conformation. The ion transport of Cs+-ions through the channels was recorded by impedance analysis.

Characterisation of the brain multidrug resistance protein (BMDP/ABCG2/BCRP) expressed at the blood-brain barrier.

The blood-brain barrier (BBB) plays the predominant role in controlling the passage of endogenous and xenobiotic substances between the circulating blood and the extracellular fluid environment of the brain. The transfer of compounds is strictly regulated by brain capillary endothelial cells (BCEC), which are interconnected to each other by well developed tight junctions, without fenestrations. Although hydrophobic molecules such as nicotine and ethanol readily cross the BBB by diffusion, the brain microvasculature shows a highly restrictive permeability to hydrophobic antitumor agents. So far, this multidrug resistance has been almost exclusively attributed to the most prominent member of the ATP-binding cassette (ABC) transporter family, P-glycoprotein located in the luminal membrane of brain capillary endothelial cells and to a minor extent to the multidrug resistance-associated proteins (MRPs). The brain multidrug resistance protein (BMDP) has recently been discovered at the porcine BBB and was shown to be highly homologous to the human breast cancer resistance protein (BCRP/ABCG2). Here, we demonstrate by northern blot and RT-PCR analysis that BMDP mRNA is more highly expressed in the capillary endothelial cells compared to other cell types of the brain. Immunocytochemistry of porcine BCEC showed a clear plasma membrane localisation of BMDP. Analysis of the total mRNA pool revealed that BMDP is more strongly expressed than P-glycoprotein and MRP1. Consistently, first transport studies indicate that active exclusion of the chemotherapeutic drug daunorubicin from the central nervous system is mediated mainly by this new transporter compared to P-glycoprotein or MRP1. Thus, we hypothesise that BMDP might play an important role in the exclusion of xenobiotics from the porcine brain.

Primary cultures of brain microvessel endothelial cells: a valid and flexible model to study drug transport through the blood–brain barrier in vitro

Studies on drug entry into the brain and permeation of the blood-brain barrier start to gain more and more importance in neuropharmaceutical research in order to develop new drugs for the therapy of central nervous system diseases. Procedures that provide quick access to permeation properties of those drugs with high throughput are difficult to achieve with animal models. Although various useful cell culture models approaching this issue have been described, results are often not comparable among each other unless determined with an equal experimental setup. Reproducibility of cell culture methods as well as corresponding findings gathered with these tools are often impeded due to the lack of details in experimental manuals. Here we present a precise manual for preparation and maintenance of porcine brain microvessel endothelial cells, serving as a culture model of the blood-brain barrier. Furthermore experimental details for blood-brain barrier transport investigations are presented. Validation of this model was carried out by determination of bioelectric properties and permeation experiments using various marker molecules reflecting paracellular and transcellular blood-brain barrier penetration. Results obtained with our model are closely resembling the in vivo-situation although astrocytes are not included. This simplification of the system is one of the major advantages towards robot derived cell cultures necessary for high throughput screening.

Mechanism of lipid-body formation in prokaryotes: how bacteria fatten up.

Neutral lipid accumulation is frequently observed in some Gram‐negative prokaryotes like Acinetobacter sp. and most actinomycetes, including the pathogenic Mycobacterium tuberculosis and antibiotic producing streptomycetes. We examined the formation  of  wax  ester‐  and  triacylglycerol  (TAG)‐bodies in Acinetobacter calcoaceticus and Rhodococcus opacus using microscopic, immunological and biophysical methods. A general model for prokaryotic lipid‐body formation is proposed, clearly differing from the current models for the formation of lipid inclusions in eukaryotes and of poly(hydroxyalkanoic acid) (PHA) inclusions in prokaryotes. Formation of lipid‐bodies starts with the docking of wax ester synthase/acyl‐CoA:diacylglycerol acyltransferase (WS/DGAT) to the cytoplasm membrane. Both, analyses of in vivo and in vitro lipid‐body synthesis, demonstrated the formation of small lipid droplets (SLDs), which remain bound to the membrane‐associated enzyme. SLDs conglomerated subsequently to membrane‐bound lipid‐prebodies which are then released into the cytoplasm. The formation of matured lipid‐bodies in the cytoplasm occurred by means of coalescence of SLDs inside the lipid prebodies, which are surrounded by a half‐unit membrane of phospholipids.

Cell adhesion monitoring using a quartz crystal microbalance: comparative analysis of different mammalian cell lines

Abstract The quartz crystal microbalance (QCM) has been widely accepted as a sensitive technique to follow adsorption processes in gas as well as in liquid environments. However, there are only a few reports about the use of this technique to monitor the attachment and spreading of mammalian cells onto a solid support in culture. Using a QCM-setup we investigated the time course of cell attachment and spreading as a function of seeding density for three widespread and frequently used cell lines (MDCK strains I and II and Swiss 3T3-fibroblasts). Results were found to be in good agreement with the geometrical properties of the individual cell types. The shifts of the resonance frequency associated with confluent cell layers on top of the quartz resonators were found to be dependent on the cell species [MDCK-I: (320±20) Hz; MDCK-II: (530±25) Hz; 3T3: (240±15) Hz] reflecting their individual influence on the shear oscillation of the resonator. These findings are discussed with respect to the basic models of materials in contact with an oscillating quartz resonator. We furthermore showed by inhibition-assays using soluble RGD-related peptides, that only specific, integrin mediated cell adhesion is detected using this QCM approach, whereas the sole presence of the cellular body in close vicinity to the resonator surface is barely detectable.

Receptor-mediated delivery of magnetic nanoparticles across the blood-brain barrier.

A brain delivery probe was prepared by covalently conjugating lactoferrin (Lf) to a poly(ethylene glycol) (PEG)-coated Fe(3)O(4) nanoparticle in order to facilitate the transport of the nanoparticles across the blood-brain barrier (BBB) by receptor-mediated transcytosis via the Lf receptor present on cerebral endothelial cells. The efficacy of the Fe(3)O(4)-Lf conjugate to cross the BBB was evaluated in vitro using a cell culture model for the blood-brain barrier as well as in vivo in SD rats. For an in vitro experiment, a well-established porcine BBB model was used based on the primary culture of cerebral capillary endothelial cells grown on filter supports, thus allowing one to follow the transfer of nanoparticles from the apical (blood) to the basolateral (brain) side. For in vivo experiments, SD rats were used as animal model to detect the passage of the nanoparticles through the BBB by MRI techniques. The results of both in vitro and in vivo experiments revealed that the Fe(3)O(4)-Lf probe exhibited an enhanced ability to cross the BBB in comparison to the PEG-coated Fe(3)O(4) nanoparticles and further suggested that the Lf-receptor-mediated transcytosis was an effective measure for delivering the nanoparticles across the BBB.

Binding of polylysine to charged bilayer membranes: molecular organization of a lipid.peptide complex.

The interaction between a positively charged peptide (poly-L-lysine) and model membranes containing charged lipids has been investigated. Conformational changes of the polypeptide as well as changes in the membrane lipid distribution were observed upon lipid-protein agglutination: 1. The strong binding of polylysine is shown directly by the use of spinlabelled polypeptide. Upon binding to phosphatidic acid a shift in the hyperfine coupling constant from 16.5 to 14.6 Oe is observed. The spectrum of the lipid-bound peptide is superimposed on the spectrum of polylysine in solution. Half of the lysine groups are bound to the charged membranes. A change in the conformation of polylysine from a random coil to a partially ordered configuration is suggested. 2. Spin labelling of the lipid component gives evidence concerning the molecular organization of a lipid mixture containing charged phosphatitid acid. Addition of polylysine induces the formation of crystalline patches of bound phosphatidic acid. 3. Excimer forming pyrene decanoic acid has been employed. Addition of positively charged polylysine (pH 9.0) to phosphatidic acid membranes increases the transition temperature of the lipid from Tt = 50 to Tt = 62 degrees C. Thus, a lipid segregation of lipid into regions of phosphatidic acid bound to the peptide which differ in their microviscosity from the surrounding membrane is induced. One lysine group binds one phosphatidic acid molecule, but only half of the phosphatidic acid is bound. 4. Direct evidence for charge induced domain formation in lipid mixtures containing phosphatidic acid is given by electron microscopy. Addition of polylysine leads to a change in the surface curvature of the bound charged lipid. The domain size is estimated from the electron micrographs. The number of domains present is dependent on both the ratio of charged to uncharged lipids as well as on the amount of polylysine added to the vesicles. The size of the domains is not dependent on membrane composition. However, the size seems to increase in a stepwise manner that is correlated with a multiple of the area covered by one polylysine molecule.