Auditory membrane vibrations: measurements at sub-angstrom levels by optical heterodyne spectroscopy.

We describe an optical technique for measurement of mechanical vibrations in the auditory organs of living animals. The technique uses light scattered from the vibrating structure and offers several new advantages. Better than 1 angstrom sensitivity, 10 micrometers spatial resolution, and > 70 decibels dynamic range are achieved. Illustrative measurements of the mechanical response of the tympanic membrane of crickets (Gryllidae) are reported.

Drug delivery using vesicles targeted to the hepatic asialoglycoprotein receptor.

We assessed the utility of liver-targeted vesicles as a drug delivery system for the treatment of liver diseases. Small, unilamellar vesicles (mean diameter, 60-80 nm) composed of dipalmitoylphosphatidylcholine, cholesterol, dipalmitoylphosphatidylglycerol and digalactosyldiacylglycerol (mol ratios, 40:40:5:15) are rapidly cleared from the blood in rats after intravenous injection. In vivo organ distribution shows that the liver is the major site of vesicle accumulation, with roughly 60-80% of the vesicle contents delivered to the liver. Isolated, perfused rat liver experiments show that the uptake is due to the hepatic asialoglycoprotein receptor, and the uptake process occurs with minimal vesicle leakage. At low doses of the vesicles, the single pass extraction by the liver is around 50%, which means that this vesicle formulation operates close to optimal efficiency as a drug delivery system to the liver. Binding of vesicles to the liver was determined to saturate at 6.5 mg total lipid/kg body weight, with a maximum steady-state turnover rate of vesicles at 37 degrees C of 79 micrograms lipid/min per kg body weight. This gives a receptor recycling time of around 80 min. We have incorporated this information into a pharmacokinetic model of vesicle distribution which quantitatively predicts the kinetics and dose dependence of vesicle uptake by the liver in vivo. This information can be used to optimize vesicle-mediated drug delivery to the liver.

Mechanism of the membrane potential sensitivity of the fluorescent membrane probe merocyanine 540.

The fluorescence and optical absorption of the membrane-staining dye merocyanine 540 (M-540) have been widely used to measure cellular transmembrane potentials. We have studied the molecular mechanisms of these optical changes by measuring the fluorescence polarization of M-540 and its response to membrane potential changes in hemispherical lipid bilayer membranes. The fluorescence responds to a potential step in two distinct time scales: a fast response with a rise time less than the instrumental capability of 6 micromilligram and a slow response with a time constant around 10(-1) s. Both response amplitudes are proportional to the amplitude of the membrane potential change and both require an asymmetrical distribution of M-540 across the membrane. The slow response is ascribed to a net change of the dye concentration in the membrane. The fast response appears to be dominated by a change in the distribution of orientations of the dye molecules in the membrane, with a concomitant perturbation of a monomer-dimer equilibrium, due to interaction of the applied electric field with the permanent molecular dipol moment of M-540. The amplitude of the fast fluorescence response is concentration dependent and can be modeled by including membrane saturation effects and the presence of a nonfluorescent dimer species in the membrane at high dye concentrations. Absorbance changes reported by other investigators are consistent with this model mechanism.

Immobilization of concanavalin A receptors during differentiation of neuroblastoma cells.

Neuroblastoma cells serve as a useful model of neuronal development because compounds such as dimethyl sulphoxide (DMSO) and dibutyryl cyclic AMP cause them to undergo a process of controlled differentiation in tissue culture, during which they can extend long processes, develop characteristic excitability mechanisms, synthesize neurotransmitters and form synapses. We have used the technique of fluorescence photobleaching recovery to study the lateral mobility of cell-surface constituents during the differentiation of neuroblastoma clone N1E-115 cells. The concanavalin A (Con A) binding sites appear as discrete patches distributed over the entire cell surface and exhibit lateral mobility in undifferentiated cells comparable with that of surface glycoproteins of other cells. After induction of differentiation, however, the vast majority of Con A binding sites become immobilized, and we present data which suggest that the mechanism of this immobilization may involve linkage to the internal actin network.

Fluorescent membrane probes and the mechanism of maintenance of cellular asymmetry in epithelia.

Individual epithelial cells are asymmetric with respect to morphology, distribution of enzymes, lipid composition, ionic permeability, and sensitivity to drugs and hormones on the apical (mucosal) versus basolateral (serosal) membranes. It has been suggested that the tight junction, which forms the morphological boundary between apical and basolateral surfaces, helps to maintain this cellular asymmetry by forming a barrier to lateral diffusion of membrane constituents across these membranes. We have directly tested this hypothesis by selectively labeling either the apical or basolateral membrane with fluorescent probes and ascertaining 1) whether the probe can diffuse laterally in the membrane, and 2) whether it can pass through the tight junction region to the side of the cell initially not labeled. Our results show that membrane-bound lectins and some lipid probes are incapable of passing through the tight junction region. The lectins are immobile on the cell surface, but the lipid probes diffuse freely in the membrane. We propose that the ability of a lipid probe to pass through the tight junction is correlated with its ability to flip-flop to the inner monolayer of the cell membrane bilayer, and that the tight junction thus forms a barrier to lipids in the outer monolayer only. Differences in diffusion rates of different lipid probes also appear to reflect different physical characteristics of the inner versus outer membrane leaflet.

Effect of bleaching light on measurements of lateral diffusion in cell membranes by the fluorescence photobleaching recovery method.

The Fc receptors for IgE on rat basophil leukemia 2H3 cells were labeled with either rhodamine- or fluorescein-conjugated IgE and then with Fab fragments of anti-IgE conjugated with the other dye. Fluorescence photobleaching recovery measurements of lateral diffusion were performed with one of the dyes before and after extensive bleaching of the other dye over an entire cell. Bleaching one dye did not affect subsequent measurements made with the other dye. We thus detect no evidence for photoinduced artifacts in fluorescence photobleaching recovery experiments.

First human studies with a high-molecular-weight iron chelator.

The release of free, reactive iron from cellular iron stores has been implicated as an important contributor to tissue damage in a variety of clinical situations, including ischemia and reperfusion injury, hemorrhagic shock, and burn injury. Deferoxamine mesylate (DFO), the only iron chelator currently approved for clinical use, is used for the treatment of iron overload, including acute iron poisoning and treatment of chronic iron overload in transfusion-dependent anemias such as beta-thalassemia. However, it is not suitable for acute care situations because of its toxicity, primarily hypotension when given at high intravenous doses, and its short plasma half-life. We have produced a high-molecular-weight iron chelator by chemically coupling DFO to hydroxyethyl starch. This novel chelator (HES-DFO) was administered to healthy male subjects by intravenous infusion over a 4-hour period. The drug was well tolerated, and signs of DFO acute toxicity were not observed. Maximum plasma chelator levels of approximately 3 mmol/L were achieved with HES-DFO, which is more than an order of magnitude higher than has been reported with injections of DFO. Drug residence time in plasma was markedly prolonged, with an initial half-life of 22 to 33 hours. Urinary iron excretion was 7.1 +/- 2.2 mg in 48 hours in the highest dose group, as compared with 0.06 +/- 0.15 mg in control subjects who received normal saline infusions. Intravenous infusion of HES-DFO is well tolerated, produces substantial and prolonged plasma chelator levels, and markedly stimulates urinary iron excretion.

Iron deprivation increases erythropoietin production in vitro, in normal subjects and patients with malignancy.

Although tissue hypoxia is the major stimulus for erythropoietin (EPO) production, serum EPO (sEPO) levels at any given Hb in iron-deficiency anaemia are relatively higher than in other anaemias. Iron chelators stimulate erythropoiesis in anaemia of chronic disease via unknown mechanisms. A recent study suggested that deferoxamine (DFO) regulates steady-state EPO RNA. Here we report that altered intracellular iron balance regulates EPO production both in vitro and in two unique clinical trials. In vitro, both iron chelation with DFO and blockade of Tf-mediated iron uptake with anti-Tf receptor antibody 42/6, stimulated EPO production in serum-deprived hepatoma cells. Conversely, iron repletion by haemin, inhibited EPO production in these cells. In clinical studies, sEPO levels rose in adult volunteers treated with DFO coupled to hydroxyethyl starch (HES-DFO) and in patients with advanced malignancy treated with anti-Tf receptor antibody 42/6, in a time- and dose-dependent manner. These studies indicate intracellular iron balance regulates EPO production in humans.