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J Eisinger

Publications and source records attributed to J Eisinger.

At least 37 records · Page 2Linked to original sources
PubMed1992

Distribution of erythrocyte free porphyrin content in erythropoietic protoporphyria.

Erythrocytes of patients suffering from erythropoietic protoporphyria (EPP) contain high levels of unchelated protoporphyrin IX (PP) molecules when they enter circulation, and the leakage of PP that leaks from the circulating cells is responsible for the patients' cutaneous photosensitivity. The level of PP in EPP blood has long been used as an indicator of the severity of the disease and is useful in its management. The present study investigates what additional information may be obtained by determining the distribution of the PP content of individual EPP red cells. Absorption and fluorescence images of fields of the dispersed and immobilized red cells from nine patients with EPP were acquired under computer control by use of an inverted fluorescence microscope equipped with a cooled slow-scan charge-coupled device camera. The distribution functions of the fluorescence emitted by individual red blood cells (IRBC) were derived by a suitable image analysis program and were converted to the distributions of the cellular PP content by relating the average value of the distributions (Iav) to the PP level of packed cells, as determined by an extraction assay. The IRBC distributions show that a small percentage of the red cells is responsible for most of the PP fluorescence, and the distributions of IRBC/Iav for the nine patients with EPP were found to be very similar. This is consistent with the leakage rate during circulation being approximately proportional to the cells' PP content.

Erythrocyte Membrane↗
PubMed1990

Resonance energy transfer from a cylindrical distribution of donors to a plane of acceptors. Location of apo-B100 protein on the human low-density lipoprotein particle.

The resonance energy transfer (RET) from a cylindrical assembly of donors to acceptors in a plane was investigated, and the dependence the average RET rate (kT) on the cylinder's size, shape, and proximity to the acceptor plane was determined. This geometry provides a model for the RET from a donor-containing protein to acceptors embedded in an associated phospholipid mono- or bilayer. The determination of kT for a series of acceptors at different levels in the phospholipid layer is shown to provide information on the protein's relationship to the phospholipid layer. Two models for the donor (D) and acceptor (A) distributions are employed: (a) The D's and A's are uniformly distributed in the cylinder and the plane, respectively, and analytical expressions for kT in terms of experimental parameters are derived. (b) The RET rates between all D, A pairs within the cylinder and in the plane are calculated and averaged for a large number of random D and A distributions. The average transfer rates obtained by the two approaches are in agreement and the width of the frequency distribution of kT for the latter provides an estimate of the error to be expected when, as is usually the case, the true D and A locations are unknown. This methodology is illustrated by analyzing RET from the 37 tryptophan residues of the apo-B100 protein to a series of pyrenylphosphatidylcholine acceptors inserted in the phospholipid monolayer of the human low-density lipoprotein particle, and it is concluded that significant portions of the protein penetrate the phospholipid layer.

Apolipoprotein B-100↗
PubMed1990

Lateral diffusivity of lipid analogue excimeric probes in dimyristoylphosphatidylcholine bilayers.

The lateral mobility of pyrenyl phospholipid probes in dimyristoylphosphatidylcholine (DMPC) vesicles was determined from the dependence of the pyrene monomeric and excimeric fluorescence yields on the molar probe ratio. The analysis of the experimental data makes use of the milling crowd model for two-dimensional diffusivity and the computer simulated random walks of probes in an array of lipids. The fluorescence yields for 1-palmitoyl-2-(1'-pyrenedecanoyl)phosphatidylcholine (py10PC) in DMPC bilayers are well fitted by the model both below and above the fluid-gel phase transition temperature (Tc) and permit the evaluation of the probe diffusion rate (f), which is the frequency with which probes take random steps of length L, the host membrane lipid-lipid spacing. The lateral diffusion coefficient is then obtained from the relationship D = fL2/4. In passing through the fluid-gel phase transition of DMPC (Tc = 24 degrees C), the lateral mobility of py10PC determined in this way decrease only moderately, while D measured by fluorescence photobleaching recovery (FPR) experiments is lowered by two or more orders of magnitude in gel phase. This difference in gel phase diffusivities is discussed and considered to be related either to (a) the diffusion length in FPR experiments being about a micrometer or over 100 times greater than that of excimeric probes (approximately 1 nm), or (b) to nonrandomicity in the distribution of the pyrenyl probes in gel phase DMPC. At 35 degrees C, in fluid DMPC vesicles, the diffusion rate is f = 1.8 x 10(8) s-1, corresponding to D = 29 microns2 s-1, which is about three times larger than the value obtained in FPR experiments. The activation energy for lateral diffusion in fluid DMPC was determined to be 8.0 kcal/mol.

Dimyristoylphosphatidylcholine↗
PubMed1990

Dipyrenylphosphatidylcholines as membrane fluidity probes. Relationship between intramolecular and intermolecular excimer formation rates.

In the intramolecular excimeric membrane probe, dipyrenylphosphatidylcholine (dipyn PC), pyrene moieties are linked to the terminal carbons of the two acyl chains, each of which contains n carbons. We show here how the probe intramolecular excimer production rate, K, may be determined from the excimer/monomer intensity ratio, rl, by making use of the fluorescence titrations of the related monopyrenyl probe, pyn PC, analyzed according to the milling crowd model. rl and the rate K of dipy10 PC in four model membrane systems were measured over a wide temperature range and both parameters are shown to be sensitive functions of the lateral fluidity of the host matrix. A model for relating the intramolecular and intermolecular excimer formation rates is proposed according to which both processes are limited by the reorientational rate of the pyrene moiety. Above the fluid-gel transition temperature, Tc, the diffusion rate (f) of the monopyrenyl probe (pyn PC) is accordingly related to K by: pE approximately K/(K + 1/2f + tau -1M), where pE is the probability of excimer formation between nearest neighbor pyn PC probes, and tau M is the monomer lifetime. Values of pE derived in this way are found to be consistent with pE values derived from the milling crowd analysis of fluorescence yield titration experiments. K for dipy10 PC in DMPC multibilayers ranges from 0.21 x 10(7) s-1 at 10 degrees C in the gel phase, to 5.7 x 10(7) s-1 at 60 degrees C in the fluid phase, whereas the lateral diffusion coefficient, D, for py10 PC in the same bilayers ranged from 8 to 34 microns2 s-1, when calculated with D = fL2/4, L being the average lipid-lipid spacing of the host membrane. Above Tc and at the same reduced temperature, (T - Tc)/Tc, both f for py10 PC, and K for dipy10 PC were found to have relative magnitudes in the order: DPPC greater than DMPC greater than POPC greater than DOPC. This and the similarity of the activation energies for f and K suggest that the rotation of the the pyrene moiety is the rate-limiting step for both the lateral mobility of py10 PC and intramolecular excimer formation in dipy10 PC.

Diffusion↗
PubMed1989

Lateral diffusion of phospholipids in the lipid surface of human low-density lipoprotein measured with a pyrenyl phospholipid probe.

Human low-density lipoprotein (LDL) was labelled with the excimeric fluorescent phospholipid analogue 1-palmitoyl-2-(1'-pyreneoctanoyl)-sn-glycero-3-phosphocholine by using phosphatidylcholine-specific transfer protein for the probe insertion. The lateral diffusivity of the probe in the phospholipid/cholesterol surface monolayer of LDL was determined from the measured dependence of the pyrene monomer fluorescence yield on probe concentration. The data were analyzed by the milling-crowd model (J. Eisinger et al. (1986) Biophys. J. 49, 987-1001] to obtain the short-range lateral diffusivity of the probe. The lateral mobility of the probe in LDL was compared to that in model lipid systems, i.e. in protein-free LDL-like lipid particles and in small unilamellar vesicles, with a phospholipid/cholesterol composition characteristic of LDL. This analysis with the probability PE = 1 for excimer production between nearest-neighbour probes gives the lower limits for f, the frequency of translational lipid--lipid exchanges of the probe of 0.62 x 10(8), 0.19 x 10(8) and 0.19 x 10(8)s-1 in LDL, LDL-like lipid particles, and small unilamellar vesicles, respectively. The lower limits for the corresponding lateral diffusion constants are 16, 5 and 5 microns 2 s-1. The results suggest that the translational mobility of phospholipid molecules in the lipid--protein surface of LDL is not constrained by the apolipoprotein B-100 moiety or the neutral lipid core of the lipoprotein. Instead, the protein moiety may perturb the lipid order with the lipid--associating peptide domains and thus fluidize the amphiphilic surface monolayer of LDL relative to the protein-free model systems. In general, lateral diffusivity of the pyrenyl phospholipid probe in LDL and the model lipid systems is comparable to the lateral mobility of lipid analogue probes in a variety of model and biological membranes.

Androgen-Binding Protein↗
PubMed1987

The lateral fluidity of erythrocyte membranes. Temperature and pressure dependence.

The pressure and temperature dependence of the lateral and rotational fluidity of erythrocyte membranes was investigated by inserting the excimeric membrane probe 1'-pyrenedodecanoic acid (PDA) into the membranes of intact cells and measuring the probe excimer formation rate and the steady-state polarization of the monomer at pressures up to 2000 atm (2 kbar). At that pressure the lateral diffusivity of PDA was found to decrease by a factor of 10 and its emission anisotropy by a factor of 5 at 22 degrees C. At atmospheric pressure, the local lateral diffusion coefficient of PDA at 2 and 33 degrees C is 1.5 and 4.3 x 10(-8) cm2 s-1, respectively. The activation energy for probe translation was found to decrease from 6 to 3 kcal M-1 in going from atmospheric pressure to 2 kbar, while the entropy decreased by approx. 15 cal M-1 K-1, indicating greater lipid order at the high pressure. The experimental data are consistent with a 'free-area' model for the membrane, analogous to the free-volume model for nonassociated liquids. The lateral diffusivity of PDA was found to be proportional to the free membrane area and linear extrapolation to zero diffusivity indicates that at atmospheric pressure, the fractional free area of the erythrocyte membrane is 6%.

Erythrocyte Membrane↗
PubMed1987

Repetitive electromyographic activity, spasmorhythmia and spasmophilia.

Spasmorhythmia (SR) can be defined as repetitive electromyographic activity lasting for longer than 4 min under conditions of cuff-induced ischemia. In a study on more than 180 'normal' subjects, this electrical anomaly was found in 21% of men and 30% of women, and was independent of age. Spontaneous variation in SR several hours or weeks apart is relatively slight, an average of 2 min, with the electrical diagnosis being confirmed by a second test in three quarters of cases. Correlations between SR and magnesium metabolism have not been proved as yet. SR has no pathological significance on its own. It assumes diagnostic importance when found in association with various vascular conditions; when found in association with a characteristic symptomatology, it may be used to support a diagnosis of latent tetany.

Adolescent↗
PubMed1986

Effects of spatial variation in membrane diffusibility and solubility on the lateral transport of membrane components.

There exist many examples of membrane components (e.g. receptors) accumulating in special domains of cell membranes. We analyze how certain variations in lateral diffusibility and solubility of the membrane would increase the efficiency of transport to these regions. A theorem is derived to show that the mean-time-of capture, tc, for particles diffusing to a trap from an annular region surrounding it, is intermediate to the tc values that correspond to the minimum and maximum diffusion coefficients that obtain in this region. An analytical solution for tc as a function of the gradient of diffusivity surrounding a trap is derived for circular geometry. Since local diffusion coefficients can be increased dramatically by reducing the concentration of intra-membrane particles and/or allowing them to form aggregates, such mechanisms could greatly enhance the diffusion-limited transport of particular membrane components to a trap (e.g. coated pit). If the trap is surrounded by an annular region in which the probe particles' partition function is increased, say, by the local segregation of certain phospholipids, tc is shown to vary inversely with the logarithm of the relative partition function. We provide some conjectural examples to illustrate the magnitude of the effects which heterogeneities in diffusibility and solubility may have in biological membranes.

Diffusion↗
PubMed1986

A milling crowd model for local and long-range obstructed lateral diffusion. Mobility of excimeric probes in the membrane of intact erythrocytes.

A new model for lateral diffusion, the milling crowd model (MC), is proposed and is used to derive the dependence of the monomeric and excimeric fluorescence yields of excimeric membrane probes on their concentration. According to the MC model, probes migrate by performing spatial exchanges with a randomly chosen nearest neighbor (lipid or probe). Only nearest neighbor probes, one of which is in the excited state, may form an excimer. The exchange frequency, and hence the local lateral diffusion coefficient, may then be determined from experiment with the aid of computer simulation of the excimer formation kinetics. The same model is also used to study the long-range lateral diffusion coefficient of probes in the presence of obstacles (e.g., membrane proteins). The dependence of the monomeric and excimeric fluorescence yields of 1-pyrene-dodecanoic acid probes on their concentration in the membranes of intact erythrocytes was measured and compared with the prediction of the MC model. The analysis yields an excimer formation rate for nearest neighbor molecules of approximately 1 X 10(7) s-1 and an exchange frequency of approximately greater than 2 X 10(7) s-1, corresponding to a local diffusion coefficient of greater than 3 X 10(-8) cm2 s-1. This value is several times larger than the long-range diffusion coefficient for a similar system measured in fluorescence photobleaching recovery experiments. The difference is explained by the fact that long-range diffusion is obstructed by dispersed membrane proteins and is therefore greatly reduced when compared to free diffusion. The dependence of the diffusion coefficient on the fractional area covered by obstacles and on their size is derived from MC simulations and is compared to those of other theories lateral diffusibility.

Diffusion↗
PubMed1986

Vitamin B6 and magnesium.

Administration of vitamin B6 at doses of 1 and 1.5 g/day, for 2-7 weeks, showed that only the high doses increased erythrocyte magnesium. Similarly, magnesium balance studies in subjects receiving 500, 1,000 and 1,500 mg/day of vitamin B6 for 8 days showed that doses of 1 g or less had no effect upon the intestinal absorption of magnesium. In view of the existence of reversible neurological complications in subjects taking 2 g of pyridoxine per day, the use of very high doses of vitamin B6 must be considered as inadvisable, even if effective. A study of the long-term effects, and upon leukocyte magnesium, of doses of 1 g or less would be desirable.

Adolescent↗
PubMed1985

Fluorometry of turbid and absorbant samples and the membrane fluidity of intact erythrocytes.

In employing intrinsic or extrinsic fluorophores in the study of whole cells, or other strongly absorbant and/or scattering samples, the measured fluorescence intensity and polarization is seriously affected by absorption and scattering within the sample cuvet. These artifacts are analyzed and simple protocols are provided for overcoming them. An expression relating attenuation of the observed emission anisotropy to sample turbidity is derived. The validity of the method is confirmed by experiments in which the emission anisotropies and fluorescence yields of membrane probes in intact erythrocytes was measured with precision. It is also shown that the rotational mobility of the membrane probe 1-phenyl-3-(2-naphthyl)-2-pyrazoline is the same for intact erythrocytes and ghosts. These protocols are particularly useful in measuring the intrinsic fluorescence yield ratio for excimeric and monomeric emission of pyrene-containing membrane probes. This provides a method for determining the local lateral mobility of excimeric probes in intact erythrocytes.

Erythrocyte Membrane↗
PubMed1985

Fluorescent cytoplasm and Heinz bodies of hemoglobin Köln erythrocytes: evidence for intracellular heme catabolism.

Hb Köln, one of the common mutant hemoglobins responsible for unstable hemoglobin disease, was found to be degraded to a fluorescent yellow pigment (FYP) in circulating erythrocytes. FYP is responsible for a strong green fluorescence observed in the cytoplasm and is particularly abundant in the Heinz bodies of Köln RBC. Front face fluorometry and fluorescence microscopy showed that Heinz bodies emit 10% to 20% of the fluorescence of RBCs. Hb-free FYP was obtained by means of a cellulose column separation of the cytoplasm or from a precipitate formed during the incubation of Köln RBC cytoplasm at 50 degrees C. The absorption and emission spectra of FYP are consistent with those of dipyrroles.

Adult↗
PubMed1984

The cytosol-membrane interface of normal and sickle erythrocytes. Effect of hemoglobin deoxygenation and sickling.

The effect of deoxygenation on the amount of hemoglobin (Hb) proximal to the membranes of intact, density-fractionated normal (AA) and sickle (SS) red cells was studied by estimating resonance energy transfer efficiencies from fluorescent probes, 12-(9-anthroyloxy)stearic acid or 2-(9-anthroyloxy)stearic acid, in the outer lipid layer to cytoplasmic hemes. For each density fraction, heme concentrations at the cytosol-membrane interface (hb) were derived from the probe decay rates for ghosts and intact cells, measured by front-face fluorometry, and compared with mean cell heme concentrations (hc). With AA cells, hb/hc varied little with cell density; a 33% drop in hb on deoxygenation is attributable to organic phosphate binding to deoxy-Hb. With oxy-SS cells, hb/hc increased with cell density to twice the values for AA cells, but SS ghosts showed no evidence of increased probe quenching by membrane-bound Hb. On deoxygenation, hb for each SS density fraction fell (reversibly) to one-third the oxy value. The finding that deoxy-HbS withdraws from the membrane bilipid layer much more than deoxy-HbA is consistent with evidence for an increased net negative charge on deoxy-HbS polymers and/or the suggestion that the cytoskeleton, readily penetrated by monomeric Hb, presents a barrier to polymeric HbS. Membrane-associated HbS is therefore thought to play an unimportant role in polymerization.

Cytosol↗
PubMed1983

Cytosol-membrane interface of human erythrocytes. A resonance energy transfer study.

The resonance energy transfer from donors embedded in the membrane of erythrocytes to the cytosol hemoglobin has been measured by comparing the donors' fluorescence decay in ghosts and in intact cells. A series of n - (9-anthroyloxy) stearic acids (n-AS) (n = 2, 6, 9, 12) and similar probes were used as donors, and their locations within the outer leaflet of the phospholipid bilayer were determined from their average efficiency of energy transfer, . The energy transfer data for several membrane probes were analyzed according to a simple semiempirical model, in which the heme acceptors are assumed to form a semiinfinite continuum beyond a plane, whose normal distance (d) from particular donors may be determined if the heme density in the cytosol boundary layer is known. The hemoglobin concentration in the erythrocytes was varied by suspending the cells in buffers of different ionic strengths. This made it possible to study the ionic strength dependence of the heme concentration averaged over the cell (h(c)), as well as that in the boundary layer (h(b)). Both level off above approximately 600 mosM, as does the ratio h(b)/h(c). By using the maximum heme concentration that can be obtained in osmotically shrunken cells as a limiting value, h(b) is estimated to be 17 mM or less, under physiological conditions; and from the measured for various probes, the distance d was found to range from 40 A for 2-AS to 31 A for 12-AS and 26 A for 9-vinyl anthracene (9-VA). It is concluded that the hydrophobic probe 9-VA is located near the center of the phospholipid bilayer and that the cytosol hemoglobin is in contact with the inner membrane surface, or nearly so. This conclusion is valid for oxy- and deoxy-hemoglobin, and is shown to be independent of several systematic errors that might arise from the simple assumptions of the model used. The steady-state fluorescence anisotropy of the probes was found to decrease as they approach the bilayer's central plane. The methodology developed here may be used to extend studies of cytosol membrane interactions in ghost systems to intact cells, and is useful in the investigation of the morphology of normal and pathological intact erythrocytes.

Cytosol↗