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.
Humans↗
Biomedical subjects
B R Ware
Publications and source records attributed to B R Ware.
Actin critical concentration optimizes at intermediate [cytochalasin B]/[actin] ratios.
Effect of cytochalasin B on actin critical concentration has been assayed using the fluorescence enhancement of pyrene-labeled actin. A peak effect of cytochalasin B on the critical concentration is observed in the presence of either 100 mM K+, or 100 mM K+ plus 2 mM Mg2+. This may result from two competing activities of cytochalasin B, one associated with its capping activity to the barbed end of actin filaments, and the other associated with its lateral binding site(s) along the filaments resulting in a severing activity of cytochalasin B.
Actins↗
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.
Actins↗
pH dependence of actin self-assembly.
Fluorescence enhancement and fluorescence photobleaching recovery have been utilized to examine actin self-assembly over the pH range 6.6-8.0. The kinetics of assembly are faster and the critical concentrations are lower at lower pH. Filament diffusion coefficients are not a function of pH, indicating that average filament lengths are not pH dependent. Although critical actin concentrations are a sensitive function of the concentrations of various cations in the medium, the relative pH dependences of critical concentrations are similar for all combinations of cations employed. The pH dependence of actin self-assembly is sufficiently great that it should be taken into account when comparing data from different reports and when relating in vitro measurements to cytoplasmic mechanisms.
Actins↗
A two-year self-assessment evaluation by students in a family medicine clerkship.
Explore the source record for details and available documents.
Clinical Clerkship↗
Rheology of G-actin solutions.
Recent reports that dilute solutions of G-actin form viscoelastic gels have been investigated using several experimental variations of three distinct physical techniques in independent laboratories. Direct measurement of storage and loss moduli were made using two rheometers of different design. Measurements of the diffusion of G-actin and of tracer particles added to G-actin solutions were carried out using dynamic light scattering and fluorescence photobleaching recovery techniques. Measurements were performed over a period of many hours, on actin solutions prepared under conditions for which anomalous gelation had been reported. All data from this investigation are consistent with the conclusion that dilute G-actin solutions behave as newtonian liquids.
Actins↗
The molecular mobility of alpha-actinin and actin in a reconstituted model of gelation.
Dictyostelium discoideum alpha-actinin (D.d. alpha-actinin) is a calcium and pH-regulated actin-binding protein that can cross-link F-actin into a gel at a submicromolar free calcium concentration and a pH less than 7 [Fechheimer, et al., 1982]. We examined mixtures of actin and D.d. alpha-actinin at four pH and calcium concentrations that exhibited various degrees of gelation or solation. The macroscopic viscosities of these mixtures were measured by falling ball viscometry (FBV) and compared to the translational diffusion coefficients measured by gaussian spot and periodic-pattern fluorescence photobleaching recovery (FPR) of both the actin filaments and D.d. alpha-actinin. A homogeneous, macroscopic gel was not composed of a static actin network. Instead, the filament diffusion coefficient decreased to approximately 65% of the control value. If the D.d. alpha-actinin concentration was increased, the solution became inhomogeneous, consisting of domains of higher actin concentration. These domains were often composed of a static actin network. The mobility of D.d. alpha-actinin consisted of a major fraction that freely diffused and a minor fraction that appeared immobile under the conditions employed. This suggested that D.d. alpha-actinin binding to the actin filaments was static over the time course of measurement (approximately 5 sec). Under solation conditions, there was no apparent interaction of actin with D.d. alpha-actinin. These results demonstrate that 1) actin filaments need not be cross-linked into an immobile, static array in order to have macroscopic properties of a gel; 2) interpretation of the rheological properties of actin:alpha-actinin gels are complicated by spatial heterogeneity of the filament concentration and mobility; and 3) a fraction of D.d. alpha-actinin binds statically to actin in undisturbed gels. The implications of these results are discussed in relation to cytoplasmic structure and contractility.
Actinin↗
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.
Actin Cytoskeleton↗
Analysis of rhodamine and fluorescein-labeled F-actin diffusion in vitro by fluorescence photobleaching recovery.
Properties of filamentous acetamidofluorescein-labeled actin and acetamidotetramethylrhodamine-labeled actin (AF and ATR-actin, respectively) were examined to resolve discrepancies in the reported translational diffusion coefficients of F-actin measured in vitro by FPR and other techniques. Using falling-ball viscometry and two independent versions of fluorescence photobleaching recovery (FPR), the present data indicate that several factors are responsible for these discrepancies. Gel filtration chromatography profoundly affects the viscosity of actin solutions and filament diffusion coefficients. ATR-actin and, to a lesser degree, AF-actin show a reduction in viscosity in proportion to the fraction labeled, presumably due to filament shortening. Actin filaments containing AF-actin or ATR-actin are susceptible to photoinduced damage, including a covalent cross-linking of actin protomers within filaments and an apparent cleavage of filaments detected by a decrease of the measured viscosity and an increase in the measured filament diffusion coefficients. Quantum yields of the two photoinduced effects are quite different. Multiple cross-links are produced relative to each photobleaching event, whereas less than 1% filament cleavage occurs. Substantial differences in the filament diffusion coefficients measured by FPR are also the result of differences in illumination geometry and sampling time. However, under controlled conditions, FPR can be used as a quantitative tool for measuring the hydrodynamic properties of actin filaments. Incremented filament shortening caused by photoinduced cleavage or incremental addition of filament capping proteins produces a continuous and approximately linear increase of filament diffusion coefficients, indicating that filaments are not associated in solution. Our results indicate that actin filaments exhibit low mobilities and it is inferred that actin filaments formed in vitro by column-purified actin, under standard conditions, are much longer than has conventionally been presumed.
Actins↗
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+.
Actins↗
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.
Actins↗
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.
Actins↗
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.
Cholestanol↗
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.
Actins↗
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.
Actins↗
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.
Acetylcholinesterase↗
Mobility of cytoplasmic and membrane-associated actin in living cells.
We have combined fluorescent analogue cytochemistry with fluorescence photobleaching recovery to measure the mobility of fluorescently labeled actin and other labeled test proteins microinjected into living amoebae. Bovine serum albumin, ovalbumin, and ribonuclease A have a cytoplasmic mobility, expressed as a diffusion coefficient, that is 1/2 to 1/3 of that observed in aqueous solution; 90% of the actin has a mobility 1/2 to 1/8 of that of G-actin in aqueous solution, and approximately equal to 10% of the actin has a mobility comparable to that of F-actin in aqueous solution. Therefore, no more than 10% of the actin in the cytoplasm of amoebae can exist as static filaments. Microinjection of phalloidin decreases the diffusion coefficient of the mobile component of cytoplasmic actin, and it also increases the low-mobility fraction to 50% but has no effect on the mobility of labeled ovalbumin. By comparing the mobility of actin in different parts of amoebae and by separating cytoplasm from plasmalemma-ectoplasm, we found the low-mobility fraction of actin to be enriched in the tail, along the plasmalemma-ectoplasm, and in contracted cytoplasm.
Actins↗
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.
Animals↗