Electrokinetic behavior of inside-out vesicles from human red cell membranes.

The electrokinetic behavior of red cell membrane vesicles of normal (ROV) and inverted (IOV) sidedness has been characterized using the laser Doppler technique of electrophoretic light scattering (ELS). At neutral pH ROV have a (approx. 25%) higher electrophoretic mobility than IOV and the two peaks can be resolved in the ELS spectrum to provide a quantitative estimate of the IOV/ROV ratio which is consistent with the ratio determined by assay of the activity of acetylcholinesterase. The ROV peak coincides with the mobility of fresh red blood cells and of resealed ghosts. Neuraminidase treatment reduces the ROV mobility by a factor of 2.6, while the IOV peak is reduced only slightly (less than 5%). Treatment with trypsin results in a single narrow ELS peak at about 60% of the mobility of ROV. Treatment of IOV with phospholipase C leaves the electrophoretic mobility unaltered, whereas treatment with phospholipase D increases their mode mobility by 22%. The mobility titration curve of IOV from pH 2 to pH 10 reveals three distinct inflection points which may be assigned to chemical groups on the cytoplasmic surface of the red cell membrane.

Effect of sterol incorporation on head group separation in liposomes.

Electrophoretic mobilities of multilamellar liposomes of varying composition have been measured to determine the effect of incorporated sterols on surface charge density. Liposomes made from mixtures of zwitterionic egg phosphatidylcholine (PC) and anionic egg phosphatidylglycerol (PG) in varying proportions were shown to have electrophoretic mobilities consistent with the anticipated surface charge density. Incorporation of cholesterol up to 50 mole per cent in the bilayer produced no detectable change in surface charge density. Similar results were obtained for lanosterol and epicoprostanol. These results are interpreted to mean that incorporation of the sterols into the bilayers produced no detectable change (less than 3%) in the spacing of charged phospholipids. It is inferred that sterols are incorporated among the fatty acyl chains of these phospholipid bilayers with little or no displacement of the head groups at the surface.

Phospholipid dependence of calcium ion effects on electrophoretic mobilities of liposomes.

Electrophoretic light scattering (ELS) and depolarization of fluorescence have been used to determine the effect of membrane fluidity on the binding of Ca2+ to liposomes. ELS was used to measure the electrophoretic mobilities of the liposomes. Fluorescence depolarization was used to determine membrane fluidity. Zero to 30 mol% phosphatidylserine (PS) was incorporated into liposomes containing, as bulk phospholipids, one of the following: dimyristoyl-phosphatidylcholine (DMPC), dipalmitoylphosphatidylcholine (DPPC), egg phosphatidylcholine (PC), or hydrogenated egg phosphatidylcholine (H egg PC). The binding of Ca2+ to the liposomes appears to be influenced by membrane fluidity. Liposomes containing bulk phospholipids whose phase transition temperature is higher than the experimental temperature exhibit enhanced binding of CA2+.

A national study of required family medicine clinical rotations.

The authors collected information from each family medicine department with a required clinical rotation. Timing of such rotations are predominantly during the third year (56%), using a combination of family practice centers and community preceptor sites. Formal didactic instructions are offered for a mean of 28 hours. This instruction is offered in combinations of introductory, weekly, and terminal didactic blocks. Most programs (68%) assign reading from an article file, and 51% develop their own examinations. Clinical evaluations represent the largest portion (61%) of final grades, with 30% coming from examinations and lesser amounts from home visits, student presentations, etc. The implications of these findings are discussed.

The office diagnosis of common intestinal parasitic diseases.

The diagnosis of parasitic infections is becoming more frequent in the ambulatory setting. This article explains collection of specimens, testing, and considerations.

Acanthamoeba profilin binding to fluorescein-labeled actins.

The binding constants of Acanthamoeba profilin to fluorescein-labeled actin from Acanthamoeba and from rabbit skeletal muscle have been determined by measuring the reduction in the actin tracer diffusion coefficients, determined by fluorescence photobleaching recovery, as a function of added profilin concentration. Data were analyzed using a two-parameter nonlinear regression analysis to determine the profilin-actin dissociation constant Kd and the profilactin diffusion coefficient, DPA. For fluorescein-labeled Acanthamoeba actin, the least-squares estimates for Kd and DPA, along with approximate single standard deviation confidence intervals, are Kd = 48 (36, 63) microM and DPA = 6.72 (6.62, 6.81) X 10(-7) cm2s-1. For fluorescein-labeled skeletal muscle actin, the corresponding values are Kd = 147 (94, 225) microM and DPA = 6.7 (6.3, 7.0) X 10(-7) cm2s-1. These dissociation constants are the first to be determined from direct physical measurement; they are in agreement with values inferred from earlier studies on the effect of profilin on the assembly of actin that had been fluorescently labeled or otherwise modified at Cys 374. These results place important restrictions on the interpretation of experiments in which fluorescently labeled actin is used as a probe of living cytoplasm or cytoplasmic extracts that include profilin.

Distinctions between mechanisms of cytochalasin D activity for Mg2+- and K+-induced actin assembly.

The fraction of assembled actin and the diffusion coefficients of filamentous and nonfilamentous species have been determined by fluorescence photobleaching recovery. For both Mg2+-induced and K+-induced actin assembly, a higher concentration of cation leads to longer filaments. Cytochalasin D reduces the fraction of actin present as assembled filaments. In the absence of Mg2+, the accompanying increase in diffusion coefficient of the filaments is of an appropriate magnitude to be accounted for by shortening of filaments as a result of net depolymerization. In the presence of Mg2+, cytochalasin D induces a dose-dependent increase in diffusion coefficient up to about a factor of 10. This increase indicates a shortening of filaments consistent with extensive filament cleavage. Under all conditions studied, the unassembled actin is present primarily as monomer.

Detection and characterization of actin monomers, oligomers, and filaments in solution by measurement of fluorescence photobleaching recovery.

Fluorescence photobleaching recovery (FPR) was measured to determine the diffusion coefficient of fluorescein-labeled G-actin in low-salt buffer. The result obtained, 7.15 +/- 0.35 X 10(-7) cm2/s, is in good agreement with that computed from the molecular weight, partial specific volume, and sedimentation coefficient, but is higher than previously obtained values. It is demonstrated from theory that at low ionic strength, the electrostatic contribution to the intrinsic viscosity leads to an overestimate of the hydrodynamic eccentricity of G-actin. Data from FPR, sedimentation, and fluorescence polarization experiments all indicate that the true low-salt form of the actin monomer has an axial ratio less than or equal to 3.0. The G-F transformation of actin was also observed by measurement of FPR during the assembly phase, in the steady state, and in the presence of ligands such as cytochalasin and aldolase. Each FPR record in general yields three data: relative proportion of rapidly and slowly diffusing actin, diffusion coefficient for the high-mobility fraction, and a mean diffusion coefficient for the low-mobility fraction. A relation between the mean low-mobility diffusion coefficient and the number-average filament length is derived and applied to the analysis of FPR data. Under typical conditions, the average filament length was much greater than 10 micron in the steady state. Cytochalasin D was found to decrease filament length and total amount of filament proportionally; total filament number was not greatly affected. In all polymerizations of G-actin, the high-mobility material observed in situ was found to be essentially monomeric actin. Relatively stable oligomers of actin were separated by fractionating G-AF-actin by gel filtration in 50 microM MgCl2 at 4 degrees C. On the basis of the diffusion coefficient, we conclude that monomer and dimer constitute the major particle types present under these conditions. Sedimentation of labeled actin polymerized in 1.0 mM MgCl2 yielded a graded supernatant that contained actin oligomers significantly larger than the monomer.

Macrophage response to concanavalin A: effect of surface crosslinking on the electrophoretic mobility distribution.

Electrophoretic light scattering (laser Doppler electrophoresis) has been employed to study the effects of concanavalin A (Con A) and succinyl-Con A on the electrophoretic mobility distribution of resident guinea pig peritoneal macrophages. Con A, a tetrameric lectin, decreases slightly the mean mobility and increases by approximately 3-fold the width of the electrophoretic mobility distribution of resident macrophages. This effect can be abolished by alpha-methyl-D-mannoside, a hapten sugar of Con A. These effects were present in both low (0.010 M) and high (physiological, 0.15 M) ionic-strength media. Since lower ionic strengths correspond to a larger Debye screening distance, these data suggest that the alterations in the electrophoretic mobility distribution are not restricted to the outer portion of the glycocalyx. Succinyl-Con A, a dimeric derivative, was found to have no effect on the mobility distribution. However, the mean mobility decreased and the width increased over 4-fold when succinyl-Con A-treated macrophages were exposed to anti-Con A. These observations indicate that cross-linking of Con A receptors is an important process in the electrokinetic alterations of the macrophage surface. These results may have important consequences for the elucidation of the details of the endocytic mechanism.

Electrokinetic properties of synaptic vesicles and synaptosomal membranes.

Using the technique of electrophoretic light scattering, we have measured the electrophoretic mobilities of synaptic vesicles and synaptosomal plasma membranes isolated from guinea-pig cerebral cortex. The electrophoretic mobility of synaptic vesicles is slightly greater than that of synaptosomal plasma membranes. Ca+2 and Mg+2 reduced the mobility of both species to the same extent at physiologically relevant concentrations (0-1 mM) and near-physiologic ionic strength. The extent of the reduction was not large (approximately 6% for synaptic vesicles in the presence of 100 mM KCl) at 1 mM divalent cation concentrations. At concentrations of approximately 2 mM and higher, Ca+2 reduced the mobility of synaptic vesicles more than did Mg/2. A similar but much smaller effect was observed in the case of synaptosomal plasma membranes. The addition of 1 mM Mg+2-ATP had no effect upon synaptic vesicle mobility either in the presence or absence of the ionophores nigericin or valinomycin. These data, together with earlier work (Siegel et al., 1978, Biophys. J. 22:341-346), demonstrate that substantial reduction of the average electrostatic surface charge density is not the most important role of divalent cations in promoting close approach of secretory granules and secretory cell membranes, and that it is certainly not the Ca+2-specific step in exocytosis.

Actin assembly by lithium ions.

The ability of Li+ to promote the assembly of actin has been compared with the more common cations used in actin assembly assays, K+, Mg2+, and Ca2+. The principal assay of actin assembly utilized was fluorescence photobleaching recovery (FPR), from which it is possible to determine the fraction of actin protomers incorporated into filaments and the average diffusion coefficients of the filaments. In addition, critical concentrations of actin over a range of concentrations of all of these cations have been determined using an assay that involves sonication and dilution of assembled actin filaments containing trace amounts of pyrene-labeled actin. The results demonstrate that Li+ is a more potent promoter of actin assembly than is K+. The more rapid assembly of actin in the presence of Li+ is attributable to an increased rate of filament elongation. Filaments assembled in equivalent concentrations of Li+ or K+ have the same diffusion coefficients, and thus presumably the same average lengths. The critical concentration of actin is about three times less in the presence of Li+ than in the presence of an equal concentration of K+. Cytochalasin D accelerates the rate of Li+-promoted actin assembly and reduces slightly the total fraction of actin assembly. However, cytochalasin D causes less shortening of filaments in the presence of Li+ than in the presence of K+ or Mg2+. By the criteria of assembly kinetics and critical concentration, Li+ is much less potent as a promoter of actin assembly than either Mg2+ or Ca2+. These results are discussed in terms of the role of electrostatic forces in the actin assembly mechanism and in terms of possible relationships to therapeutic and toxicity mechanisms for Li+.

Velocity distributions of the streaming protoplasm in Nitella flexilis.

Laser light is Doppler-shifted in frequency by the streaming endoplasm of living cells of Nitella flexilis. The frequency spectrum of the scattered light can be interpreted as the histogram of velocities within the organism, with the exception of the intense low-frequency portion of the spectrum. We demonstrate that the lowest-frequency component is the result of amplitude modulation of the scattered light by the array of chloroplasts in the cell. Measurement of the streaming endoplasm in a photobleached "window" region allows correction of the frequency distribution for the modulation component. The complete velocity histogram for the streaming endoplasm is calculated directly from the corrected frequency distribution. Measurements of vacuolar and endoplasmic motions show that the tonoplast, the membrane separating the vacuole and the endoplasm, seems to be flowing along with the endoplasm and vacuolar sap. Placing the cell in medium containing ATP in concentrations greater than 10(-3) M greatly increases the contribution of low velocities to the velocity histogram. Cytochalasin B at high dosages (10-50 mug/ml) does not noticably change the shape of the velocity histogram, while at low dosages (1 mug/ml) there is an increase in the contribution of low velocities to the velocity histogram. Colchicine in high concentrations (1%) has no observable effect on the velocity histogram.

Electrokinetic response of granulocytes to concanavalin A and succinyl-concanavalin A.

Electrophoretic light scattering has been used to study the effects of concanavalin A (Con A) and succinyl-Con A on the electrophoretic mobility distribution of resident guinea-pig peritoneal eosinophils and human peripheral blood polymorphonuclear leukocytes. In both cell types, incubation with Con A (a tetrameric lectin) decreases slightly the mean mobility and increases substantially the width of the electrophoretic mobility distribution. These effects can be abolished by alpha-methyl-D-mannoside, a hapten sugar of Con A. Succinyl Con A, a dimeric derivative, was found to have no effect on the mobility distribution. These results are strikingly similar to our previous report of the response of the resident guinea-pig macrophage (19), suggesting possible parallels in the endocytic mechanisms of these cell types.

Assembly of Acanthamoeba actin in the presence of Acanthamoeba profilin measured by fluorescence photobleaching recovery.

Assembly of Acanthamoeba actin, of which trace quantities had been labeled with 5-(iodoacetamido)-fluorescein, was quantified using the modulation detection method of fluorescence photobleaching recovery (FPR). This technique permits explicit determination of the fraction of labeled actin incorporated into filaments and the translational diffusion coefficients of the filaments, from which filament length can be calculated. Addition of Acanthamoeba profilin in molar ratios to actin of about 1.1:1 and 2.3:1 retarded the initial kinetics of assembly (induced by addition of 2mM Mg+2) and reduced the fraction of actin incorporated into filaments. The diffusion coefficients of filaments formed were greatly changed by the presence of profilin at short times, but the differences became increasingly smaller at longer times. After 26 hr. the filaments formed in 1.1:1 profilin were about 12% shorter and in 2.3:1 profilin were about 20% shorter than filaments formed by actin alone under the same conditions.

A study of protoplasmic streaming in Nitella by laser Doppler spectroscopy.

Laser light scattered from particles in the streaming protoplasm of a living cell is shifted in frequency by the Doppler effect. The spectrum of the scattered light can be measured and interpreted to infer details of the velocity distribution in the protoplasm. We have developed this approach to study the protoplasmic streaming in the fresh-water alga Nitella. Our results indicate a characteristic flow pattern to which diffusion makes a negligible contribution. No difference in the velocity of particles of different size is indicated. The streaming velocity linearly with temperature with a supraoptimal temperature of 34 degrees C, and the velocity distribution becomes narrower at high temperatures. The protoplasmic streaming can be inhibited by laser light, and this effect has been used to study the photoresponse of the algae. Using beam diameters of about 50 mum, we have shown that the inhibition is very local, becoming minimal at a displacement of about 200 mum in the upstream direction and 400 mum in the downstream direction. Prolonged exposure produces a bleached area free of chloroplasts, which is three orders of magnitude less sensitive to photoinhibition.

Electrophoretic mobilities and diffusion coefficients of hemoglobin at high pH.

Diffusion studies by photon correlation of scattered laser light confirm the dissociation of the tetrameric form of human carboxyhemoglobin to dimers above pH 10 and provide new estimates of the subunit dissociation equilibrium constants in this pH range. Electrophoretic light-scattering experiments under the same conditions reveal that the electrophoretic mobilities of tetramers and dimers are indistinguishable to within instrumental resolution (ca. 7% in these experiments). The data imply an increase of the electrical charge on the dimer of at least 2.8 to 4.4 net negative charges upon dissociation. Mechanisms for the accumulation of negative charge by the dimer upon dissociation of the tetramer are proposed.

Actin filament capping and cleaving activity of cytochalasins B, D, E, and H.

The concentration dependences of the activities of cytochalasin B, D, E, and H in capping and cleaving actin filaments have been assayed using fluorescence photobleaching recovery. Filament capping was detected by the increase in mobile G-actin. Cytochalasin D (CD) showed the strongest filament capping activity, with an apparent dissociation constant from filament ends of 50 nM. The order of capping activity was CD greater than CH greater than CE much greater than CB. Filament cleavage was detected by the increase in the diffusion coefficients of actin filaments. By this criterion the order of filament cleavage activity was CD, CE greater than CH much greater than CB. Cytochalasin B shows some activity in cleavage of filaments over a concentration range (0-100 microM) at which it shows no appreciable capping activity. This activity, together with results from other groups, is interpreted to mean that CB binds to protomers within the filament, but not to the barbed end. The reversal of activities for CH and CE, combined with the activity profile of CB, constitute the strongest evidence to date that there is more than one cytochalasin binding site on the actin molecule.

Actin assembly activity of cytochalasins and cytochalasin analogs assayed using fluorescence photobleaching recovery.

The effects on actin self-assembly of 9 common cytochalasins and 9 synthetic analogs have been assayed using fluorescence photobleaching recovery (FPR). The specific assembly activities of cytochalasins determined by this assay are (i) reduction of the fraction of actin molecules incorporated into filaments; (ii) increase of the steady-state diffusion coefficients of filaments, from which filaments shortening may be inferred; and (iii) acceleration of the initial rate of assembly. Of the compounds studied, only cytochalasin D shows strong activity of all three types. The range of activities shown by other compounds indicates clearly that these three activity types are distinct and independent. Inspection of the molecular structures of these 18 compounds for correlation of structure and activity reveals that the three different activities depend on distinct structural features. The Mg2+ dependence of filament-shortening activity by certain cytochalasins may be explained by the Mg2+ chelating ability of two suitably positioned oxygen atoms on the convex face of the bicyclic isoindolone system. Inhibition of filament elongation may involve very specific, high-affinity cytochalasin interactions at a binding site on terminal actin molecules, while accelerating activity may occur by weaker, less specific binding interactions of cytochalasins with monomeric actin.