Monitoring of single and double lipid membrane formation with high spatiotemporal resolution using evanescent light scattering microscopy.

Formation and quality of single solid supported lipid membranes and double lipid membranes were investigated with single vesicle resolution using label-free evanescence light scattering microscopy (EvSM). For the formation of double lipid membranes we made use of electrostatic interaction between charged lipids and oppositely charged cations.

Interaction of virions with membrane glycolipids.

Cellular membranes contain various lipids including glycolipids (GLs). The hydrophilic head groups of GLs extend from the membrane into the aqueous environment outside the cell where they act as recognition sites for specific interactions. The first steps of interaction of virions with cells often include contacts with GLs. To clarify the details of such contacts, we have used the total internal reflection fluorescence microscopy to explore the interaction of individual unlabelled virus-like particles (or, more specifically, norovirus protein capsids), which are firmly bound to a lipid bilayer, and fluorescent vesicles containing glycosphingolipids (these lipids form a subclass of GLs). The corresponding binding kinetics were earlier found to be kinetically limited, while the detachment kinetics were logarithmic over a wide range of time. Here, the detachment rate is observed to dramatically decrease with increasing concentration of glycosphingolipids from 1% to 8%. This effect has been analytically explained by using a generic model describing the statistics of bonds in the contact area between a virion and a lipid membrane. Among other factors, the model takes the formation of GL domains into account. Our analysis indicates that in the system under consideration, such domains, if present, have a characteristic size smaller than the contact area between the vesicle and the virus-like particle.

Interaction of single viruslike particles with vesicles containing glycosphingolipids.

Glycosphingolipids are involved in the first steps of virus-cell interaction, where they mediate specific recognition of the host cell membrane. We have employed total-internal-reflection fluorescence microscopy to explore the interaction kinetics between individual unlabeled noroviruslike particles, which are attached to a glycosphingolipid-containing lipid bilayer, and fluorescent vesicles containing different types and concentrations of glycosphingolipids. Under association equilibrium, the vesicle-binding rate is found to be kinetically limited, yielding information on the corresponding activation energy. The dissociation kinetics are logarithmic over a wide range of time. The latter is explained by the vesicle-size-related distribution of the dissociation activation energy. The biological, pharmaceutical, and diagnostic relevance of the study is briefly discussed.

A multitechnique study of liposome adsorption on Au and lipid bilayer formation on SiO2.

We have used a new setup for parallel quartz crystal microbalance with dissipation (QCM-D) and surface plasmon resonance (SPR) measurements to measure the detailed kinetics of vesicle-to-bilayer transformation on SiO2 and vesicle adsorption on Au, respectively. The combination of SPR and QCM-D, complemented by atomic force microscopy measurements, has enabled a complete, time-resolved separation of vesicle and bilayer coverages, and thus, for the first time, allowed precise quantification of the critical surface coverage of vesicles needed for rupture. We furthermore demonstrate and quantify a previously undetected vesicle-size- and concentration-dependent loss of lipid material during the later stages of the process.

Lateral mobility of tethered vesicle-DNA assemblies.

Supported lipid membranes are particularly attractive for use in biochemical assays because of their resistance to nonspecific adsorption and their unique ability to host transmembrane proteins. Although ideal for use in many surface-based detection techniques, supported bilayers can make the incorporation of proteins problematic due to the steric constraints of the underlying substrate. A recently developed strategy overcomes this obstacle by tethering liposomes to supported lipid bilayers via cholesterol-tagged DNA. Due to the fluidity of the bilayer, the vesicle assemblies exhibited significant lateral mobility. The corresponding diffusion coefficients were then investigated using fluorescence recovery after photobleaching (FRAP). The diffusivity was neither sensitive to the size of the vesicles nor to the length of the DNA tether. However, changing from single cholesterol tethers to double cholesterol tethers caused a decrease in the diffusivity of the assemblies by a factor of 3. Perhaps even more notable was the fact that single cholesterol-DNA without vesicles diffused 6 times faster than the corresponding assemblies. Double cholesterol-DNA diffused 11 times faster. This discrepancy is believed to arise from the fact that each vesicle is tethered to the bilayer by multiple DNA pairs.

Pregnancy outcomes in urban teenagers.

OBJECTIVES: To compare pregnancy outcomes for teenagers with those for older gravidas. METHODS: A retrospective case control study was undertaken to compare teenagers who delivered between January 1996 and October 1999 at a public urban hospital with a group of older gravidas. RESULTS: Young pregnant teenagers were more likely to be nulliparous. They weighed less and gained less in pregnancy. More teen pregnancies occurred among Hispanics than other ethnic groups. The younger the teenager, the more likely for her infant to be of low or very low birth weight or growth restricted. There were fewer postmature deliveries, macrosomic fetuses and cesarean deliveries in young gravidas. Perinatal mortality was unaffected by maternal age. CONCLUSIONS: This investigation demonstrates that, while pregnancy outcomes in teenagers have improved in recent years relative to historical patterns, teenagers face continuing problems requiring special attention by care givers.

Temperature dependence of formation of a supported phospholipid bilayer from vesicles on SiO2.

Adsorption of egg-phosphatidylcholine vesicles and bilayer formation on a SiO2 surface was investigated in the temperature range 278-303 K using the quartz crystal microbalance-dissipation technique. The critical coverage for the vesicle-->bilayer transition is found to decrease with increasing temperature. The temperature dependence of the time-scale characterizing this transition can be represented in the Arrhenius form. Higher temperatures produce a bilayer with fewer trapped, nonruptured vesicles.

Simulation of two-dimensional streptavidin crystallization.

We present lattice Monte Carlo simulations of the growth of streptavidin islands at a biotinylated lipid layer. The model employed takes into account attractive anisotropic lateral interactions between streptavidin tetramers. With a minimal set of interactions, we reproduce the formation of rectangular islands experimentally observed at pH > or = 9.0. Specifically, we analyze two scenarios of the island growth. First, if streptavidin is rapidly adsorbed at t = 0 (stepwise coverage change without ongoing adsorption), the average linear island size is found to grow according to the Lifshitz-Slyozov law, R proportional to t(1/3). Second, if the island growth occurs in parallel with streptavidin adsorption limited by diffusion in the solution, the Lifshitz-Slyozov law is also applicable, but only at the late stage, when the streptavidin coverage is appreciable.

Variations in coupled water, viscoelastic properties, and film thickness of a Mefp-1 protein film during adsorption and cross-linking: a quartz crystal microbalance with dissipation monitoring, ellipsometry, and surface plasmon resonance study.

We have measured the time-resolved adsorption kinetics of the mussel adhesive protein (Mefp-1) on a nonpolar, methyl-terminated (thiolated) gold surface, using three independent techniques: quartz crystal microbalance with dissipation monitoring (QCM-D), surface plasmon resonance, and ellipsometry. The QCM-D and ellipsometry data shows that, after adsorption to saturation of Mefp-1, cross-linking of the protein layer using NaIO4 transforms it from an extended (approximately 20 nm), water-rich, and hydrogel-like state to a much thinner (approximately 5 nm), compact, and less water-rich state. Furthermore, we show how quantitative data about the thickness, shear elastic modulus, and shear viscosity of the protein film can be obtained with the QCM-D technique, even beyond the Sauerbrey regime, if frequency (f) and energy dissipation (D) measurements measured at multiple harmonics are combined with theoretical simulations using a Voight-based viscoelastic model. The modeling result was confirmed by substituting H2O for D2O. As expected, the D2O substitution does not influence the actual adsorption behavior, but resulted in expected differences in the estimated effective density and shear viscosity. These results provide new insight and understanding about the adsorption kinetics and crosslinking behavior of Mefp-1. They also demonstrate how the above three techniques complement each other for biomolecule adsorption studies.

Measurement of the dynamics of exocytosis and vesicle retrieval at cell populations using a quartz crystal microbalance.

The quartz crystal microbalance-dissipation technique (QCM-D) is used in two different measurement strategies to monitor the mass change and rigidity of populations of excitable cells during exocytosis and subsequent retrieval of dense-core vesicles. Two cell lines, NG 108-15 and PC 12, were grown to confluence on piezoelectric quartz crystals and were examined separately to demonstrate differences in release and retrieval with cells of different morphology, size, and number of dense-core vesicles. Stimulating the cells to exocytosis with media containing an elevated potassium concentration resulted in an increase in the frequency response corresponding to loss of mass from the cells owing to release of vesicles. In Ca2+-free media, the response was completely abolished. The amplitude and peak area in the frequency response corresponding to mass change with stimulated release was larger for PC 12 cells than for NG 108-15 cells, whereas the initial rate constants for the frequency responses were similar. The data suggest (1) that a greater number and larger size of vesicles in PC 12 cells results in a greater amount of release from these cells vs NG 108-15 cells, (2) the recycling of vesicles utilizes similar fusion/retrieval mechanisms in both cell types, (3) that the control of excess retrieval might be related to the number and size of released vesicles, and (4) that measured retrieval has a rapid onset, masking exocytosis and implying a rapid retrieval mechanism in the early stages of release. These results demonstrate that measurements of complex dynamic processes relating to dense-core vesicle release and retrieval can be simultaneously accomplished using the QCM-D technique.

Comparison of surface plasmon resonance and quartz crystal microbalance in the study of whole blood and plasma coagulation.

The coagulation of blood plasma and whole blood was studied with a surface plasmon resonance (SPR) based device and a quartz crystal microbalance instrument with energy dissipation detection (QCM-D). The SPR and QCM-D response signals were similar in shape but differing in time scales, reflecting differences in detection mechanisms. The QCM-D response time was longer than SPR, as a physical coupling of the sample to the substrate is required for molecules to be detected by the QCM-method. Change of sample properties within the evanescent field is sufficient for detection with SPR. Both the SPR signals and the QCM-D frequency and dissipation shifts showed dependency on concentrations of coagulation activator and sensitivity to heparin additions. The ratio of dissipation to frequency shifts, commonly considered to reflect viscoelastic properties of the sample, varied with the concentration of activator in blood plasma but not in whole blood. Additions of heparin to the thromboplastin activated whole blood sample, however, made the ratio variation reoccur. Implications of these observations for the understanding of the blood coagulation processes as well as the potential of the two methods in the clinic and in research are discussed.

Measurements Using the Quartz Crystal Microbalance Technique of Ferritin Monolayers on Methyl-Thiolated Gold: Dependence of Energy Dissipation and Saturation Coverage on Salt Concentration.

The adsorption kinetics of ferritin as a function of ionic strength has been studied with a new quartz crystal microbalance technique, allowing simultaneous measurement of the frequency shift (proportional to the mass uptake under certain conditions) and of changes in the energy dissipation caused by the adlayer. The measurements were performed with methyl-terminated (hydrophobic) thiol-covered gold surfaces, at pH 7.0 and ionic strengths in the range 1-200 mM KCl. The saturation uptake increases rapidly with increasing ionic strength in the range 20-50 mM and is then independent of ionic strength at >100 mM. The dissipation factor reveals, in the low coverage regime, distinct differences in the adlayer properties at low and high ionic strength, respectively. These results are briefly discussed in terms of the screening properties of the solvent and its influence on the protein-protein interaction in solution and on the surface. Copyright 1998 Academic Press.

Structural changes in hemoglobin during adsorption to solid surfaces: effects of pH, ionic strength, and ligand binding.

We have studied the adsorption of two structurally similar forms of hemoglobin (met-Hb and HbCO) to a hydrophobic self-assembled methyl-terminated thiol monolayer on a gold surface, by using a Quartz Crystal Microbalance (QCM) technique. This technique allows time-resolved simultaneous measurements of changes in frequency (f) (c.f. mass) and energy dissipation (D) (c.f. rigidity/viscoelastic properties) of the QCM during the adsorption process, which makes it possible to investigate the viscoelastic properties of the different protein layers during the adsorption process. Below the isoelectric points of both met-Hb and HbCO, the DeltaD vs. Deltaf graphs displayed two phases with significantly different slopes, which indicates two states of the adsorbed proteins with different visco-elastic properties. The slope of the first phase was smaller than that of the second phase, which indicates that the first phase was associated with binding of a more rigidly attached, presumably denatured protein layer, whereas the second phase was associated with formation of a second layer of more loosely bound proteins. This second layer desorbed, e.g., upon reduction of Fe3+ of adsorbed met-Hb and subsequent binding of carbon monoxide (CO) forming HbCO. Thus, the results suggest that the adsorbed proteins in the second layer were in a native-like state. This information could only be obtained from simultaneous, time-resolved measurements of changes in both D and f, demonstrating that the QCM technique provides unique information about the mechanisms of protein adsorption to solid surfaces.

Simultaneous frequency and dissipation factor QCM measurements of biomolecular adsorption and cell adhesion.

We have measured the energy dissipation of the quartz crystal microbalance (QCM), operating in the liquid phase, when mono- or multi-layers of biomolecules and biofilms form on the QCM electrode (with a time resolution of ca. 1 s). Examples are taken from protein adsorption, lipid vesicle adsorption and cell adhesion studies. Our results show that even very thin (a few nm) biofilms dissipate a significant amount of energy owing to the QCM oscillation. Various mechanisms for this energy dissipation are discussed. Three main contributions to the measured increase in energy dissipation are considered. (i) A viscoelastic porous structure (the biofilm) that is strained during oscillation, (ii) trapped liquid that moves between or in and out of the pores due to the deformation of the film and (iii) the load from the bulk liquid which increases the strain of the film. These mechanisms are, in reality, not entirely separable, rather, they constitute an effective viscoelastic load. The biofilms can therefore not be considered rigidly coupled to the QCM oscillation. It is further shown theoretically that viscoelastic layers with thicknesses comparable to the biofilms studied in this work can induce energy dissipation of the same magnitude as the measured ones.

QCM Operation in Liquids: An Explanation of Measured Variations in Frequency and Q Factor with Liquid Conductivity.

Recently, several reports have shown that when one side of a quartz crystal microbalance (QCM) is exposed to a liquid, the parallel (but not the series) resonant frequency is influenced by the conductivity and dielectric constant of the liquid. The effect is still controversial and constitutes a serious complication in many applications of the QCM in liquid environments. One suggestion has been that acoustically induced surface charges couple to charged species in the conducting liquid. To explore this effect, we have measured the parallel and the series mode resonance frequencies, and the corresponding Q factors, for a QCM with one side facing a liquid. These four quantities have all been measured versus liquid conductivity, using a recently developed experimental setup. It allows the simultaneous measurement of the resonant frequency and the Q factor of an oscillating quartz crystal, intermittently disconnected from the driving circuit. Based on these results, a simple model together with an equivalent circuit for a quartz crystal exposed to a liquid is presented. The analysis shows that it is not necessary to infer the existence of surface charges (or other microscopic phenomena such as electrical double layers) to account for the influence of the liquid's electrical properties on the resonant frequency. Our results show that the contacting conductive liquid, in effect, enlarges the electrode area on the liquid side and thereby changes the parallel resonant frequency. By proper design of the QCM measurement, perturbing effects due to the liquid's electrical properties can be circumvented.

Suramin, a protein kinase C inhibitor, impairs hepatic regeneration.

Previously we have shown that partial hepatectomy (PH) or exposure of the liver to the mitogen prolactin induces activation of hepatic protein kinase C (PKC). Here, we used suramin, an antitrypanosomal and chemotherapeutic drug which inhibits that enzyme, as a probe of PKC signal transduction in the regenerative response after PH in the rat. Suramin was administered i.p. in nonhepatotoxic doses of 20 to 160 mg/kg 14 days prior to PH. Three measures of hepatic DNA synthesis or cell division, thymidine kinase activity, [3H]thymidine incorporation, and mitotic index were inhibited in a dose-dependent fashion. Baseline PKC activity, in both the cytosolic and particulate fractions, was unchanged by suramin. After PH, PKC activation, signalled by an increase in activity in the particulate fraction, was observed in control rats at 30 and 60 min. However, rats which had previously received suramin demonstrated dose-dependent inhibition of PKC activation. Suramin is known to also disrupt the binding of certain growth factors to their receptors. But if inhibition of PKC activation were conferred by interference with growth factor-receptor binding by suramin, then the generation of diacylglycerol, the second messenger for PKC activation, should likewise be impaired. However, we observed that the diacylglycerol mass generated at 15, 30, and 45 min after PH was not altered by suramin pretreatment. We conclude that the diminution in DNA synthesis after PH by suramin is likely the consequence of direct inhibition of PKC, suggesting that PKC activation is an important, perhaps obligatory, signal transduction event in liver regeneration.

Light-induced voltage changes associated with electron and proton transfer in photosystem II core complexes reconstituted in phospholipid monolayers.

We have measured light-induced voltage changes (electrogenic events) in photosystem II (PSII) core complexes oriented in phospholipid monolayers. These events are compared to those measured in the functionally and structurally closely related reaction centers from the photosynthetic bacterium Rhodobacter sphaeroides. In both systems we observed a rapid (< 100 ns) light-induced increase in voltage associated with charge separation. In PSII reaction centers it was followed by a decrease (decay) of approximately 14% of the charge-separation voltage and a time constant of approximately 500 microseconds. In bacterial reaction centers this decay was approximately 9% of the charge-separation voltage, and the time constant was approximately 200 microseconds. The decay was presumably associated with a structural change. In bacterial reaction centers, in the presence of excess water-soluble cytochrome c2+, it was followed by a slower increase of approximately 30% of the charge-separation voltage, associated with electron transfer from the cytochrome to the oxidized donor, P+. In PSII reaction centers, after the decay the voltage remained on the same level for > or = 0.5 s. In PSII reaction centers the electron transfer Q-AQB-->QA Q-B contributed with an electrogenicity of < or = 5% of that of the charge separation. In bacterial reaction centers this electrogenicity was < or = 2% of the charge-separation electrogenicity. Proton transfer to Q2-B in PSII reaction centers contributed with approximately 5% of the charge-separation voltage, which is approximately a factor of three smaller than that observed in bacterial reaction centers.

A group III twintron encoding a maturase-like gene excises through lariat intermediates.

The 1605 bp intron 4 of the Euglena gracilis chloroplast psbC gene was characterized as a group III twintron composed of an internal 1503 nt group III intron with an open reading frame of 1374 nt (ycf13, 458 amino acids), and an external group III intron of 102 nt. Twintron excision proceeds by a sequential splicing pathway. The splicing of the internal and external group III introns occurs via lariat intermediates. Branch sites were mapped by primer extension RNA sequencing. The unpaired adenosines in domains VI of the internal and external introns are covalently linked to the 5' nucleotide of the intron via 2'-5' phosphodiester bonds. This bond is susceptible to hydrolysis by the debranching activity of the HeLa nuclear S100 fraction. The internal intron and presumptive ycf13 mRNA accumulates primarily as a linear RNA, although a lariat precursor can also be detected. The ycf13 gene encodes a maturase-like protein that may be involved in group III intron metabolism.