Aqueous Space Research Articles

Formation and stability of the dispersed particles composed of retinoic acid, sesame oil and phosphatidylcholine

All trans-retinoic acid (RA) was dispersed by sonication with soybean phosphatidylcholine (PC). The particle size in the dispersion was increased to 240 nm up to the RA mol fraction range ( X RA) of 0.4. At X RA=0.5, the RA/PC mixture was difficult to disperse and the macroscopic oil/water phase separation was observed. On the other hand, by the addition of sesame oil (SO) to RA (molar ratio of RA:SO=1:1), stable aqueous dispersions (diameter: 40–80 nm) were obtained in the mol fraction range RA and SO mixture ( X M) of 0.1–0.8. In order to clarify these dispersal mechanism, the dispersed particles were characterized and the interaction among RA, SO and PC was investigated using several physicochemical techniques. The trapped aqueous volume inside the RA/PC particles was determined using the aqueous space marker, calcein and it was increased with the addition of RA into small unilamellar vesicles of PC. On the other hand, that of RA/SO/PC particles was decreased remarkably with increase in X M and the decline in the fraction of vesicular particles was also confirmed by fluorescence quenching of N-dansylhexadecylamine in the PC membrane by the addition of the quencher CuSO 4. These results indicate that the interaction of RA with PC bilayers and the structure of RA/PC mixture will be changed by the addition of SO.

Different import pathways through the mitochondrial intermembrane space for inner membrane proteins.

Earlier work on the protein import system of yeast mitochondria has identified two soluble 70 kDa protein complexes in the intermembrane space. One complex contains the essential proteins Tim9p and Tim10p and mediates transport of cytosolically-made metabolite carrier proteins from the outer to the inner membrane. The other complex contains the non-essential proteins Tim8p and Tim13p as well as loosely associated Tim9p; its function was unclear, but it interacted structurally or functionally with the Tim9p-Tim10p complex. We now show that the two 70 kDa complexes each mediate the import of a different subset of integral inner membrane proteins and that they can transfer these proteins to one of three different membrane insertion sites: the TIM22 complex, the TIM23 complex or an as yet uncharacterized insertion site. Yeast mitochondria thus use multiple pathways for escorting hydrophobic inner membrane proteins across the aqueous intermembrane space.

Reverse Cholesterol Transport and Atherosclerosis Regression

Cholesterol is the major component of atherosclerotic plaque. Cholesterol accumulation within atherosclerotic plaque occurs when cholesterol influx into the arterial wall (from apoB-containing lipoproteins) exceeds cholesterol efflux. Increased influx of cholesterol into the arterial wall is accompanied by an increased influx of monocytes/macrophages,1 which take up oxidized and aggregated LDL and store the cholesterol as esters. Whereas parenchymal cells maintain cholesterol balance by downregulating de novo cholesterol synthesis and LDL receptor expression, macrophages continue to take up cholesterol from apoB-containing lipoproteins via pathways that are not subject to sterol-mediated feedback control. Current strategies to reduce coronary heart disease (CHD) are aimed primarily at reducing the influx of cholesterol into the arterial wall by lowering plasma LDL cholesterol concentrations. Aggressive lowering of plasma LDL levels reduces mortality from CHD,2 but protection is not complete, and in a significant proportion of patients, plasma LDL concentrations cannot be lowered to a level that would be predicted to halt the progression of disease. As a consequence, there is great interest in strategies aimed at enhancing cholesterol efflux from the arterial wall and promoting its transport to the liver for excretion. Cholesterol that is synthesized in extrahepatic tissues or acquired from lipoproteins is returned to the liver for excretion in a process called reverse cholesterol transport.3 The initial step in reverse cholesterol transport is thought to be efflux of cholesterol from cell membranes to acceptor particles in the interstitial fluid. Two models have been proposed with regard to the movement of cholesterol from plasma membrane to acceptor particles. In the first, the aqueous diffusion model, cholesterol molecules spontaneously desorb from cell membranes and are then incorporated into acceptor particles after traversing the intervening aqueous space by diffusion.4 Phospholipid vesicles, phospholipid/albumin complexes, and triglyceride/phospholipid emulsions efficiently remove cholesterol from cells via this …

Compartmentalization of the erythrocyte membrane by the membrane skeleton: intercompartmental hop diffusion of band 3.

Recent developments in microscopic instrumentation and probes have allowed the observation and manipulation of the movement of membrane proteins and lipids in the plasma membrane at the level of single molecules. These experiments are performed by tracking small colloidal gold particles attached to specific membrane proteins and lipids (single particle tracking) (Jacobson et al., 1995 ; Sheets et al., 1995 ; Kusumi and Sako, 1996 ; Saxton and Jacobson, 1997 ) or by dragging particle–protein complexes along the surface of the plasma membrane using laser tweezers (also termed an “optical trap” or OT) (Edidin et al., 1991 ; Kusumi and Sako, 1996 ; Kusumi et al., 1998 ). In optimal cases, a single protein molecule is attached to a 40-nm-diameter colloidal gold probe (Tomishige et al., 1998 ). These single molecule experiments have demonstrated that most membrane-spanning proteins do not undergo simple diffusion, but that a significant number are locally slowed or stopped, some are temporarily confined, and others are transported unidirectionally (Kusumi et al., 1993 ; Sheets et al., 1997 ; Sako et al., 1998 ; Simson et al., 1998 ). Previously, these processes had not been observed, because the methods used, e.g., fluorescence recovery after photobleaching, recorded the ensemble-averaged behavior of a large number of molecules. The underlying cellular mechanisms that induce nonrandom diffusion have remained largely unknown; however, we and others found that properties of the membrane skeleton network fundamentally affect the movement and distribution of certain membrane proteins, such as transferrin receptor, α2-macroglobulin receptor, MHC class I molecules, E-cadherin, and band 3, through corralling and binding effects imposed by the membrane skeleton network (Edidin et al., 1991 ; Luna and Hitt, 1992 ; Kusumi et al., 1993 ; Sako and Kusumi, 1994 , 1995 ; Sako et al., 1998 ; Tomishige et al., 1998 ). The results are particularly clear with band 3 in human erythrocyte ghost membranes, where the specimen consists of a lipid bilayer and its underlying membrane skeleton, which has been biochemically and biophysically well characterized (Golan, 1989 ; Bennett, 1990 ; Bennett and Gilligan, 1993 ; Mohandas and Evans, 1994 ). The results obtained by these studies support a “membrane skeleton fence model” in which the cytoplasmic portion of a membrane protein collides with the membrane skeletal “meshwork” because of steric hindrance, thus resulting in the temporal confinement of the protein within the mesh (compartment, Figure ​Figure1A).1A). Membrane proteins escape from these compartments and hop to adjacent ones because of the dynamic properties of the membrane skeleton. We predict that time-dependent fluctuations in the distance between the membrane and the skeleton are large or the membrane–skeleton network connections form and break continuously, as expected in a chemical equilibrium, providing membrane proteins with the opportunity to pass through the mesh barrier. In the case of band 3, macroscopic diffusion within the erythrocyte membrane occurs as the result of a series of such intercompartmental hops. Figure 1 (A) The model to explain the confined diffusion of band 3 in the membrane (the membrane skeleton fence model). (B) Movement of gold particles attached to band 3 in the erythrocyte membrane, observed with a temporal resolution of 8 ms (4 times greater ... The scientific content of this work has been published previously (Tomishige et al., 1998 ), and the purpose of the present essay is to give the reader a glimpse of the raw data from this study, as recorded by both normal and high-speed video microscopy. We hope the video sequences attached to this article will help the viewing audience develop a feel for how band 3 interacts with the membrane skeleton and undergoes intercompartmental hop diffusion within the erythrocyte ghost membrane.

Formation and structure of stably dispersed particles composed of retinal with dipalmitoylphosphatidylcholine: coexistence of emulsion particles with bilayer vesicles

In order to develop an intravenous formulation of all- trans-retinal (vitamin A aldehyde, VAA) for the treatment of night blindness, VAA and dipalmitoylphosphatidylcholine (DPPC) were sonicated and the dispersions in the VAA mole fraction range of 0.1–0.7 were stable at room temperature for 3 days. In order to clarify the dispersal mechanism, the dispersed particles were characterized and the interaction between VAA and DPPC was investigated using several physicochemical techniques. Dynamic light scattering measurements showed that the diameter of the dispersed particles was 50–70 nm. A limited amount of VAA is incorporated into DPPC bilayer membranes (approximately 5 mole%). The trapped aqueous volume inside the particles was determined fluorometrically using the aqueous space marker calcein and the volume in the VAA/DPPC particles was decreased remarkably with the addition of VAA into small unilamellar vesicles of DPPC. The decline in the fraction of vesicular particles was also confirmed by fluorescence quenching of N-dansylhexadecylamine in the DPPC membrane by the addition of the quencher CuSO 4. These results indicate that the excess VAA separated from the DPPC bilayers is stabilized as emulsion particles by the DPPC surface monolayer. The monolayer–bilayer equilibrium of VAA/DPPC mixtures was estimated by measurement of spreading and collapse pressures. The results showed that the coexistence of emulsion particles (surface monolayer of DPPC+ core of VAA) with vesicular particles (bilayer) was critically important for the formation of the stably dispersed particles of the lipid mixture.

The action of flufenamic acid and other nonsteroidal anti-inflammatories on sulfate transport in the isolated perfused rat liver

The influence of flufenamic acid and other nonsteroidal anti-inflammatories on sulfate transport in the liver was investigated. The experimental system was the isolated perfused rat liver. Perfusion was accomplished in an open, nonrecirculating system. The perfusion fluid was Krebs/Henseleit-bicarbonate buffer (pH 7.4), saturated with a mixture of oxygen and carbon dioxide (95:5) by means of a membrane oxygenator and heated to 37°C. Sulfate transport (equilibrium exchange) was measured by employing the multiple-indicator dilution technique with simultaneous injection (impulse input) of [ 35S]sulfate, [ 3H]sucrose (indicator for the distribution of the sinusoidal transit times), and [ 3H]water (indicator for the total aqueous space). Analysis was accomplished by means of a space-distributed variable transit time model. Flufenamic acid and other anti-inflammatories inhibited sulfate transport in the liver. For a concentration of 100 μM, the following decreasing series of potency could be established: flufenamic acid (53.4 ± 2.9%) > niflumic acid (41.1 ± 1.4%) > mefenamic acid (35.6 ± 3.3%) > piroxicam (16.6 ± 1.9%) > naproxen (13.5 ± 8.4)%) ≅ nimesulide (11.6 ± 5.8%). Inhibition of sulfate transport by flufenamic acid was clearly concentration dependent; 250 μM flufenamic acid produced more than 95% inhibition. Flufenamic acid in the range between 50 and 250 μM did not affect the mean transit times of tritiated water ( t̄ water) and [ 3H]sucrose ( t̄ suc), the same applying to all other anti-inflammatory agents (100 μM) tested in this work. This means that these agents do not affect vascular and cellular spaces, even when present at high concentrations. The ratio of the intra- to extracellular sulfate concentrations ([C] i/[C] e), generally between 0.4 and 0.5 under control conditions, was affected only by 250 μM flufenamic acid and 100 μM niflumic acid. In the first case, this phenomenon is possibly due to the high degree of transport inhibition (more than 95%), which does not allow a uniform tracer distribution over the whole cellular space during a single passage through the liver. The degree of inhibition of sulfate transport by 100 μM flufenamic acid was a function of the concentration of nontracer sulfate. With sulfate in the range between 1.2 and 25 mM, the inhibition degree increased linearly with the concentration. In the presence of flufenamic acid, the saturation curve of equilibrium exchange showed a substrate inhibition-like phenomenon, which was absent in the control curve. As inhibitors of sulfate transport in hepatocytes, flufenamic and niflumic acids are less active than in erythrocytes by a factor of 10 2. This observation is most probably indicative of structural differences between the hepatic sulfate carrier and the anion carrier of erythrocytes. It is unlikely that the action of flufenamic acid and its analogs on sulfate transport is a consequence of energy metabolism inhibition. Nimesulide is as active as flufenamic or niflumic acid in inhibiting energy metabolism but considerably less efficient as an inhibitor of sulfate transport. Our results as well as literature data reveal that the interactions of the nonsteroidal anti-inflammatories with the liver membranes and intracellular structures are ample and complex. Even at high concentrations, however, these interactions are not so intense as to change the vascular and cellular spaces.

Interaction of α-Tocopherol and Soybean Oil with Phosphatidylcholine and Their Formation of Small Dispersed Particles

In this study, α-tocopherol (α-T) was dispersed by cosonication with soybean phosphatidylcholine (PC). The particle size in the dispersion was increased to 250 nm up to the α-T mole fraction (XT) 0.4. AtXT= 0.5, the α-T/PC mixture was difficult to disperse and the macroscopic oil/water phase separation was observed. By the addition of soybean oil (SO) to α-T (molar ratio α-T:SO = 1:1), stable aqueous dispersions (diameter 50–70 nm) were obtained in the mole fraction range for the α-T and SO mixture (XM) 0.1–0.8. In order to clarify the dispersal mechanism, the dispersed particles were characterized and the interaction among α-T, SO, and PC was investigated using several physicochemical techniques. The trapped aqueous volume inside the α-T/PC particles was determined using the aqueous space marker calcein, and this volume was increased with the addition of α-T into small unilamellar vesicles of PC. The trapped aqueous volume of α-T/SO/PC particles was decreased remarkably with an increase inXM, and the decline in the fraction of vesicular particles was also confirmed by fluorescence quenching ofN-dansylhexadecylamine in the PC membrane by the addition of the quencher CuSO4. These results indicate that the interaction of α-T with PC bilayers and the structure of the α-T/PC mixture will be changed by the addition of SO.

Interaction of retinol with dipalmitoylphosphatidylcholine and their formation of small dispersed particles

Stable aqueous dispersions of all- trans-retinol (vitamin A, VA) were obtained by sonication with dipalmitoylphosphatidylcholine (DPPC) in the VA mole fraction range 0.1–0.7. In order to clarify the dispersal mechanism, the dispersed particles were characterized and the interaction between VA and DPPC was investigated using several physicochemical techniques. Dynamic light scattering measurements showed that the diameter of the dispersed particles was 50–70 nm. A limited amount of VA was incorporated into DPPC bilayer membranes (approximately 5 mol%). The trapped aqueous volume inside the particles was determined fluorometrically using the aqueous space marker calcein and the volume in the VA/DPPC particles was decreased markedly with the addition of VA into small unilamellar vesicles of DPPC. The decline in the fraction of vesicular particles was also confirmed by fluorescence quenching of N-dansylhexadecylamine in the DPPC membrane by the addition of the quencher CuSO 4. These results indicate that the excess VA separated from the DPPC bilayers is stabilized as emulsion particles by the DPPC surface monolayer.

Permeability of large unilamellar digalactosyldiacylglycerol vesicles for protons and glucose – influence of α-tocopherol, β-carotene, zeaxanthin and cholesterol

The permeability of large unilamellar vesicles formed from digalactosyldiacylglycerol for glucose and protons was measured. The vesicle composition was modified by addition of different terpenoids: α-tocopherol, cholesterol, zeaxanthin and β-carotene. The digalactosyldiacylglycerol species composition was dominated by the species 18:2/18:2 and 18:1/18:2. Using the self-quenching properties of the fluorescent probe 6-carboxyfluorescein trapped in the aqueous space of the vesicles, the permeability for glucose was determined with a glucose gradient of 800 mM. The calculated permeability coefficient for glucose was in the range of 4.85.10 –10 to 1.12.10 –9 cm.s –1. For proton permeability measurements, the pH-sensitive fluorescent dye pyranine was used. The proton permeability was measured with a pH gradient of 0.6 pH units from 7.0 to 7.6 with valinomycin present to dissipate any diffusion potential across the membrane. The permeability coefficients for protons were in the range of 3.5.10 –8 to 1.0.10 –7 cm.s –1. Digalactosyldiacylglycerol vesicles with 5 % α-tocopherol or 10 % cholesterol or 2 % zeaxanthin reduced the permeability for protons, the two latter significantly as compared to digalactosyldiacylglycerol vesicles. α-Tocopherol (5 %) decreased the permeability for glucose remarkably and so did cholesterol (10 %). β-Carotene (< than 1 %) and zeaxanthin (2 %) in galactolipid vesicles, however, increased the permeability. The significance of these results is discussed in relation to the physiological functions of galactolipids and terpenoids in chloroplast membranes.

The influenza haemagglutinin-induced fusion cascade: effects of target membrane permeability changes.

To define the stages in influenza haemagglutinin (HA)-mediated fusion the kinetics of fusion between cell pairs consisting of single influenza HA-expressing cells and single erythrocytes (RBC) which had been labelled with both a fluorescent lipid (Dil) in the membrane and a fluorescent solute (calcein) in the aqueous space have been monitored. It is shown that release of solute from the target cell occurs, following the formation of the hemi-fusion diaphragm. These results are discussed in terms of a model in which fusion peptide insertion into the target membrane induces lipid stalks, which results in the formation of a hemifusion diaphragm and a fusion pore. Bilayer expansion due to overproduction of these stalks can give rise to collateral damage of target membranes.

Interaction of Soybean Oil with Phosphatidylcholine and Their Formation of Small Dispersed Particles

Stable aqueous dispersions of soybean oil (SO) were obtained by cosonication with dipalmitoylphosphatidylcholine (DPPC) in the SO mole fraction range 0.1–0.8. To clarify the dispersal mechanism, the dispersed particles were characterized, and the interaction between SO and DPPC was investigated using several physicochemical techniques. Dynamic light scattering (DLS) measurements showed that the diameter of the dispersed particles was 40–60 nm. The trapped aqueous volume inside the particles was determined fluorometrically using the aqueous space marker calcein. The trapped volume in the SO/DPPC particles decreased remarkably with the addition of SO into small unilamellar vesicles of DPPC. The decline in fraction of vesicular particles was also confirmed by fluorescence quenching of N-dansylhexadecylamine in the DPPC membrane by the addition of the quencher CuSO4. These results indicate that the excess SO separated from the DPPC bilayers is stabilized as emulsion particles by the DPPC surface monolayer. Monolayer-bilayer equilibrium of SO/DPPC mixtures was estimated by measurement of spreading and collapse pressures. The results showed that the coexistence of emulsion particles (surface monolayer of DPPC + core of SO) with vesicular particles (bilayer) was critically important for the formation of stably dispersed particles of the lipid mixture.

Interaction of sesame oil with soybean phosphatidylcholine and their formation of small dispersed particles

Stable aqueous dispersions of sesame oil (SO) were obtained by co-sonication with soybean phosphatidylcholine (PC) in the SO mole fraction range of 0.1-0.8. In order to clarify the dispersal mechanism, the dispersed particles were characterized and the interaction of SO with PC was investigated using several physicochemical techniques. Dynamic light scattering measurements showed that the diameter of the dispersed particles was 40-60nm. The trapped aqueous volume inside the particles was determined fluorometrically using the aqueous space marker, calcein. The trapped volume in the SO/PC particles decreased remarkably with the addtion of SO intosmall unilamellar vesicles of PC. The decline in fraction of vesicular particles was also confirmed by fluorescence quenching of N-dansylhexadecylamine in the PC membrane by the addition of the quencher CuSO4. These results indicate that the excess SO separated from the PC bilayers is stabilized as emulsion particles by the PC surface monolayer. Monolayer-bilayer equilibrium of SO/PC mixtures was estimated by measurements of spreading and collapse pressures. The results showed that the coexistence of emulsion particles (surface monolayer of PC+ core of SO) with vesicular particles (bilayer) was critically important for the formation of stably dispersed particles of the lipid mixture.

Tim9p, an essential partner subunit of Tim10p for the import of mitochondrial carrier proteins

Tim10p, a protein of the yeast mitochondrial intermembrane space, was shown previously to be essential for the import of multispanning carrier proteins from the cytoplasm into the inner membrane. We now identify Tim9p, another essential component of this import pathway. Most of Tim9p is associated with Tim10p in a soluble 70 kDa complex. Tim9p and Tim10p co-purify in successive chromatographic fractionations and co-immunoprecipitated with each other. Tim9p can be cross-linked to a partly translocated carrier protein. A small fraction of Tim9p is bound to the outer face of the inner membrane in a 300 kDa complex whose other subunits include Tim54p, Tim22p, Tim12p and Tim10p. The sequence of Tim9p is 25% identical to that of Tim10p and Tim12p. A Ser67-->Cys67 mutation in Tim9p suppresses the temperature-sensitive growth defect of tim10-1 and tim12-1 mutants. Tim9p is a new subunit of the TIM machinery that guides hydrophobic inner membrane proteins across the aqueous intermembrane space.

A simple in vitro model to study the release kinetics of liposome encapsulated material

A simple in vitro model was developed to study the release kinetics of liposome encapsulated material in the presence of biologic components. Liposomes were embedded in an agarose gel (bottom layer) formed in a glass vial and separated from the receptor compartment buffer by a second layer of agarose gel (top layer). To follow the release of liposomal contents, aqueous space markers differing in molecular weight (from 205 Dalton to 17 500 Dalton) were encapsulated. The isotonic buffer in the receptor was completely changed at various time points and the amount of marker released from the agarose matrix containing the liposomes into the receptor medium determined. The release of non-encapsulated markers from the gel followed a time 0.5 relationship with about 75% of a 17 500 Dalton protein being released from the matrix in 48 h. In the same period, about 7% of the intact liposomes added to the agarose gel appeared in the receptor phase. The release of calcein from various liposome compositions including: (A) egg phosphatidylcholine (EPC)/egg phosphatidylglycerol (EPG) 9:1, (B) dioleoylphosphatidylethanolamine (DOPE)/cholesterylhemisuccinate (CHEMS) 2:1, and (C) dioleoylphosphatidylglycerol (DOPC)/dioleoylphosphatidylglycerol (DOPG) 2:1 was measured. Components of the biological milieu such as serum proteins and calcium influenced release of encapsulated material. This in vitro model is a convenient and reproducible system that permits the study of the release of high molecular weight molecules such as proteins from liposomal formulations in the presence of serum. It may find applications with respect to release of proteins from a variety of colloidal drug delivery systems.

Metabolic effects and distribution space of flufenamic acid in the isolated perfused rat liver

The following aspects were investigated in the present work: (a) the action of flufenamic acid on hepatic metabolism (oxygen uptake, glycolysis, gluconeogenesis, uricogenesis and glycogenolysis), (b) the action of flufenamic acid on the cellular adenine nucleotide levels, and (c) the transport and distribution space of flufenamic acid in the liver parenchyma. The experimental system was the isolated perfused rat liver. Perfusion was accomplished in an open, non-recirculating system. The perfusion fluid was Krebs/Henseleit-bicarbonate buffer (pH 7.4), saturated with a mixture of oxygen and carbon dioxide (95:5) by means of a membrane oxygenator and heated to 37°C. The distribution space of flufenamic acid was measured by means of the multiple-indicator dilution technique with constant infusion (step input) of [ 3H]water plus flufenamic acid. The results of the present work indicate that the metabolic effects of flufenamic acid are the consequence of an uncoupling of oxidative phosphorylation, a conclusion based on the following observations: (a) flufenamic acid increased oxygen uptake, a common property of all uncouplers; (b) the drug also increased glycolysis and glycogenolysis in livers from fed rats (these are expected compensatory phenomena for the decreased mitochondrial ATP formation); (c) flufenamic acid inhibited glucose production from fructose, an energy-dependent process; (d) the cellular ATP levels were decreased by flufenamic acid whereas the AMP levels were increased; and (e) the total adenine nucleotide content was decreased by flufenamic acid and uric acid production was stimulated. Indicator-dilution experiments with flufenamic acid revealed that this substance undergoes flow-limited distribution in the liver and that its apparent distribution space greatly exceeds the aqueous space of the liver. Flufenamic acid changed its behaviour when the portal concentration was increased from 25 to 50 μM. At 25 μM the initial upslope of the outflow profile clearly preceded that of all other concentrations. From the trend of the curves obtained with 50, 100 and 250 μM, one would expect an initial upslope situated at the right of the 50- μM curve. Furthermore, the time of appearance of flufenamic acid in the outflowing perfusate was practically the same irrespective of the portal concentration. For theoretical reasons one would expect progressively longer appearance times when the portal concentration was decreased. It is possible that the amount of flufenamic acid bound to the cell membranes during the early stages of the infusion produced changes that enabled these structures to bind a larger quantity of the drug than originally possible.

Methodologies in the Study of Cell–Cell Fusion

The process of membrane fusion has been profitably studied by fusing cells that express fusion proteins on their surfaces to the membranes of target cells. Primary methods for monitoring the occurrence of fusion between cells are measurement of formation of heterokaryons, measurement of activation of reporter genes, measurement of transfer of lipidic and aqueous fluorescent dyes, and electrophysiological recording of fusion pores. Fluorescence and electrical methods have been well developed for fusion of a nucleated cell expressing viral fusion proteins to red blood cell targets. These techniques are now being extended to the study of fusion between two nucleated cells. Microscopic observation of spread of fluorescent dyes from one cell to another is a sensitive and convenient means of detecting fusion on the level of single events. In such studies, both the membrane and the aqueous continuities that occur as a result of fusion can be measured in the same experiment. By following spread of aqueous dyes of different sizes from one cell to another, the growth of a fusion pore can also be followed. By labeling cells with fluorescent probes, a state of hemifusion can be identified if probes in outer membrane leaflets transfer but probes in inner leaflets or aqueous spaces do not. Electrical measurements—both capacitance and double-whole-cell voltage-clamp techniques—are the most sensitive methods yet developed for detecting the formation of pores and for quantifying their growth. These powerful single-event methodologies should be directly applicable to further advances in expressing nonviral fusion proteins on cell surfaces.

Cooperative coupling and role of heme a in the proton pump of heme-copper oxidases

In the last few years, evidence has accumulated supporting the applicability of the cooperative model of proton pumps in cytochrome systems, vectorial Bohr mechanism, to heme-copper oxidases. The vectorial Bohr mechanism is based on short- and long-range protonmotive cooperative effects linked to redox transitions of the metal centers. The crystal structure of oxidized and reduced bovine-heart cytochrome c oxidase reveals, upon reduction, the occurrence of long-range conformational changes in subunit 1 of the oxidase. Analysis of the crystal structure of cytochrome c oxidase shows the existence of hydrogen-bonded networks of amino acid residues which could undergo redox-linked pK shifts resulting in transmembrane proton translocation. Our group has identified four proteolytic groups undergoing reversible redox-linked pK shifts. Two groups result in being linked to redox transitions of heme a 3. One group is apparently linked to Cu B. The fourth group is linked to oxido-reduction of heme a. We have shown that the proton transfer resulting from the redox Bohr effects linked to heme a and Cu B in the bovine oxidase displays membrane vectorial asymmetry, i.e., protons are taken up from the inner aqueous space (N), upon reduction, and released in the external space (P), upon oxidation of the metals. This direction of proton uptake and release is just what is expected from the vectorial Bohr mechanism. The group linked to heme a, which can transfer up to 0.9 H +/e at pHs around neutrality, can provide the major contribution to the proton pump. It is proposed that translocation of pumped protons, linked to electron flow through heme a, utilizes a channel (channel D) which extends from a conserved aspartate at the N entrance to a conserved glutamate located between heme a and the binuclear center. The carboxylic group of this glutamic acid, after having delivered, upon electron flow through heme a, pumped protons towards the P phase, once reprotonated from the N phase, moves to deliver, subsequently, to the binuclear center chemical protons consumed in the conservation of the peroxy to ferryl and of the latter to the oxy intermediate in the redox cycle. Site-directed mutagenesis of protolytic residues in subunit 1 of the aa 3-600 quinol oxidase of Bacillus subtilis to non-polar residues revealed that the conserved Lys 304 is critical for the proton pumping activity of the oxidase. Crystal structures of cytochrome c oxidase show that this lysine is at the N entrance of a channel which translocates the protons consumed for the production of the peroxy intermediate. Inhibition of this pathway, by replacement of the lysine, short-circuits protons from channel D to the binuclear center, where they are utilized in the chemistry of oxygen reduction.

The development of IL-2 conjugated liposomes for therapeutic purposes

A unique immunoliposome has been developed as a drug delivery vehicle for immunotherapy. Human recombinant interleukin-2 (IL-2) has been chemically coupled to the external surface of small unilamellar vesicles (SUVs) containing methotrexate as a candidate immunosuppresive agent in order to specifically direct the drug-bearing liposome to activated T-cells expressing the high affinity IL-2 receptor. This drug delivery system is designed to deliver an immunosuppressive agent to those cells that actively participate in disorders such as graft rejection without delivering an effective but potentially toxic drug to all cells of the immune system as well as other healthy tissues. IL-2 was chemically modified with succinimidyl 4-[ p-maleidophenyl butyrate](SMPB) while the receptor binding domain on IL-2 was protected by monoclonal anti-IL-2 bound to Protein A-Silica Gel. The antibody recognizes the receptor binding domain of the IL-2 molecule. The IL-2 was derivatized with S-succinimidyl-S-thioacetate (SATA) in order to add an acetyl thioester group to the lipid and create the complex. The derivatized lipid (SATA-PE) was then part of the liposome formulation containing DSPC:cholesterol: SATA-PE at a mole ratio of 1.5:1.0:0.26. SMPB-IL-2 was covalently coupled to the external surface of the SUV after deacetylation of the thioester moiety at pH 7.4 in PBS. Liposomes prepared by sonication or extrusion had an average diameter of 46–50 nm. SUV-IL-2 bound to the high affinity IL-2 receptor as measured by competitive binding assays and Scatchard analysis using 111InCl 2-loaded liposomes The preparation exhibited a binding constant of 30 pM, consistent with values for free IL-2 cited in the literature. SUV IL-2 could be used as the sole source of IL-2 for the murine CTLL-2 T-cell line or for human mitogen-activated PBLs. The presence of IL-2 coupled to the surface was absolutely required for delivery of the drug to the cell. When methotrexate was encapsulated within the internal aqueous space, receptor-mediated endocytosis led to the inhibition of proliferation due to delivery of MTX to the cytoplasm of the cell. More than 90% of the methotrexate was retained within the liposome during storage over a 24-h period at 4°C. This immunoliposome represents a new class of cell specific immunoliposomes whose entry into the cell is controlled by a cell surface receptor.

Rapid diffusion of green fluorescent protein in the mitochondrial matrix.

It is thought that the high protein density in the mitochondrial matrix results in severely restricted solute diffusion and metabolite channeling from one enzyme to another without free aqueous-phase diffusion. To test this hypothesis, we measured the diffusion of green fluorescent protein (GFP) expressed in the mitochondrial matrix of fibroblast, liver, skeletal muscle, and epithelial cell lines. Spot photobleaching of GFP with a 100x objective (0.8-micron spot diam) gave half-times for fluorescence recovery of 15-19 ms with >90% of the GFP mobile. As predicted for aqueous-phase diffusion in a confined compartment, fluorescence recovery was slowed or abolished by increased laser spot size or bleach time, and by paraformaldehyde fixation. Quantitative analysis of bleach data using a mathematical model of matrix diffusion gave GFP diffusion coefficients of 2-3 x 10(-7) cm2/s, only three to fourfold less than that for GFP diffusion in water. In contrast, little recovery was found for bleaching of GFP in fusion with subunits of the fatty acid beta-oxidation multienzyme complex that are normally present in the matrix. Measurement of the rotation of unconjugated GFP by time-resolved anisotropy gave a rotational correlation time of 23.3 +/- 1 ns, similar to that of 20 ns for GFP rotation in water. A rapid rotational correlation time of 325 ps was also found for a small fluorescent probe (BCECF, approximately 0.5 kD) in the matrix of isolated liver mitochondria. The rapid and unrestricted diffusion of solutes in the mitochondrial matrix suggests that metabolite channeling may not be required to overcome diffusive barriers. We propose that the clustering of matrix enzymes in membrane-associated complexes might serve to establish a relatively uncrowded aqueous space in which solutes can freely diffuse.

Import of mitochondrial carriers mediated by essential proteins of the intermembrane space.

In order to reach the inner membrane of the mitochondrion, multispanning carrier proteins must cross the aqueous intermembrane space. Two essential proteins of that space, Tim10p and Tim12p, were shown to mediate import of multispanning carriers into the inner membrane. Both proteins formed a complex with the inner membrane protein Tim22p. Tim10p readily dissociated from the complex and was required to transport carrier precursors across the outer membrane; Tim12p was firmly bound to Tim22p and mediated the insertion of carriers into the inner membrane. Neither protein was required for protein import into the other mitochondrial compartments. Both proteins may function as intermembrane space chaperones for the highly insoluble carrier proteins.