The Surface Charge of Tritrichomonas foetus

The surface charge of Tritrichomonas foetus was evaluated by means of the binding of colloidal iron hydroxide particles at pH 1.8 and cationized ferritin particles at pH 7.2 to the cell surface, as visualized by electron microscopy and by direct measurements of the electrophoretic mobility (EPM), of cells suspended in solutions of different ionic strength and pH. At pH 7.2, T. foetus has a negative surface charge with a mean EPM of ?1.03 μmμs^?1μV^?1μcm. At lower pH, there is a decrease in the negative surface charge with an isoelectric point at pH 1.2. At higher pH (> 9.0), there is an increase in the surface charge reaching an EPM of ?2.5 μmμs^?1μV^?1μcm. These results indicate that the surface of T. foetus contains both negatively and positively charged dissociating groups. Binding of colloidal iron hydroxide and cationized ferritin particles throughout the cell surface of the protozoon was observed. Treatment of T. foetus with neuraminidase or trypsin reduced significantly the EPM of the cells. Enzyme-treated cells recovered their normal EPM when incubated for 6 h in fresh culture medium by a process that is inhibited by puromycin.     

Investigation of the connection between the electrophoretic mobility (EPM) of microorganisms and their capability of metal uptake

The electrophoretic mobility of microorganisms (EPM) as a measure of their electric surface charge was determined as a function of different milieu conditions with the aid of the “Parmoquant 2” cell electrophoresis apparatus manufactured by CARL ZEISS Jena. The object of these researches was to examine the influence of the electric surface charge of microorganisms on their metal loading capacity. The results show a direct correlation between the electric surface charge or the EPM of microorganisms and their maximum metal loading capacity. Cells with a high negative surface also posses a high metal binding capacity. On the other side only a negligible metal uptake can be observed at the isoelectric point of the microorganisms (EPM = 0). The method of cell electrophoresis proved suitable to analyze complex interactions between microorganisms and heavy metal ions.     

Calcium-dependent process in reduction of cell surface charge after x-irradiation.

The electrophoretic mobility (EPM) of rat erythrocytes and cultured melanoma cells decreased with time after X-irradiation in the presence of calcium at concentrations higher than 10 (-5) M. At 37 degrees C, the presence of calcium for the first 20 min of exposure was suffcient to induce the EPM reduction, and Ca 2+ administration subsequent to Ca 2+ -free incubation for 30 min following irradiation had no effect on EPM. At lower temperatures, from 10 down to 20 degrees C however, the effect of calcium on the reduction of EPM decreased drastically. If the cells were kept Ca 2+ -inonophore A23187 also induced to decrease in EPM only in the presence of Ca 2+. These results revealed the transitory existence of membrane condition reactive to extracellular Ca 2+ immediately after X-irradiation, which can be postponed at low temperatures. The reduction of EPM by Ca 2+ -ionophore might suggest that the influx of Ca 2+ is a step in the reduction of EPM after X-irradiation.     

Effect of ultra-low dose of ionizing radiation on the surface potential of erythrocytes

Effect of ionizing radiation in ultralow dose (5 microGy) on responses of erythrocyte electrophoretic motility (EPM) as a result of adrenoreceptor ligands binding (0.01-100 microM) has been investigated. The opposite directional EPM responses to agonists (adrenaline, isoprenaline) and antagonist (propranolol) of beta-adrenoreceptors was shown. At that, EPM response to the radiation coincides both with the direction and value of acting the beta-adrenoreceptor agonists depressing EPM. The EPM response to a combined action of beta-adrenoreceptors antagonist, propranolol (10 microM), and ionizing radiation is additive. The above listed is capable to evidence about the essential role of adrenoreceptors at formation of erythrocyte membrane surface charge under action of ionizing radiation in ultralow doses.     

Electric field-driven transformations of a supported model biological membrane--an electrochemical and neutron reflectivity study

A mixed bilayer of cholesterol and dimyristoylphosphatidylcholine has been formed on a gold-coated block of quartz by fusion of small unilamellar vesicles. The formation of this bilayer lipid membrane on a conductive surface allowed us to study the influence of the support's surface charge on the structure and hydration of the bilayer lipid membrane. We have employed electrochemical measurements and the specular reflection of neutrons to measure the thickness and water content in the bilayer lipid membrane as a function of the charge on the support's surface. When the surface charge density is close to zero, the lipid vesicles fuse directly on the surface to form a bilayer with a small number of defects and hence small water content. When the support's surface is negatively charged the film swells and incorporates water. When the charge density is more negative than −8 μC cm⁻², the bilayer starts to detach from the metal surface. However, it remains in a close proximity to the metal electrode, being suspended on a thin cushion of the electrolyte. The field-driven transformations of the bilayer lead to significant changes in the film thicknesses. At charge densities more negative than −20 μC cm⁻², the bilayer is ~37 Å thick and this number is comparable to the thickness determined for hydrated multilayers of dimyristoylphosphatidylcholine from x-ray diffraction experiments. The thickness of the bilayer decreases at smaller charge densities to become equal to ~26 Å at zero charge. This result indicates that the tilt of the acyl chains with respect to the bilayer normal changes from ~35° to 59° by moving from high negative charges (and potentials) to zero charge on the metal.     

Surface charge of trypanosoma cruzi. Binding of cationized ferritin and measurement of cellular electrophoretic mobility.

The surface charge of epimastigote and trypomastigote forms of Trypanosoma cruzi was evaluated by means of binding of cationized ferritin to the cell surface as visualized by electron microscopy, and by direct measurements of the cellular microelectrophoretic mobility (EPM). Epimastigote forms had a mean EPM of -0.52 micrometer-s-1-V-1-cm and were lightly labeled with cationized ferritin. In contrast, bloodstream trypomastigotes had a much higher EPM (-1.14), and the surface was heavily labeled with cationized ferritin. When trypomastigotes from staionary phase cultures were isolated on DEAE cellulose columns, the mean EPM was found to be significantly lower (-0.63), and labeling with cationized ferritin decreased. With a mixed population containing epimastigote, trypomastigote, and intermediate forms, EPM values ranging between -0.70 to -1.14 were found. From these observations we conclude that there is a definite increase in negative surface charge during development from epi- to trypomastigote forms of T. cruzi.     

Melittin-induced changes in thylakoid membranes: particle electrophoresis and light scattering study

Thylakoids were used as a model system to evaluate the effect of bee venom peptide melittin (Mt) on membrane surface charge. At neutral pH, thylakoid membrane surfaces carry excess negative electrical charge. Mt strongly altered the electrophoretic mobility (EPM) of 'low-salt' thylakoids and did not significantly change the EPM of 'high-salt' thylakoids. Mt increased the primary ionic-exchange processes across the 'low-salt' thylakoid membranes, while it did not affect those of 'high-salt' thylakoids. Mt decreased the proton gradient generation on the membranes at both ionic strengths, but it affected more strongly the 'high-salt' than that of 'low-salt' thylakoids. The primary photochemical activity of photosystem II, estimated by the ratio Fv/Fm, was not influenced by the low Mt concentrations. It decreased only when chloroplasts had been incubated with higher Mt concentrations and this effect was better expressed in 'low-salt' than in 'high-salt' thylakoid membranes.     

The electrophoretic mobility of gram-negative and gram-positive bacteria: an electrokinetic analysis.

The electrophoretic mobility (EPM) of a variety of Gram-negative and Gram-positive bacteria was measured with a Penkem S3000 analyser. Under standard growth conditions and neutral pH all cells displayed a negative EPM. The polysaccharide capsules of Escherichia coli strains K1, K5, K29 and K30 generated the highest EPM; to a lesser and varying degree O-antigens with charged groups and core lipopolysaccharides also contribute to the net EPM. Very little negative EPM was measured in suspension cultures of the gliding bacterium Cytophaga U67. No difference in the EPM was observed between rapidly growing and stationary-phase E. coli B. De-energization of the cell membranes by carbonyl cyanide m-chlorophenylhydrazone (CCCP) did not affect the EPM of wild-type and deep rough mutants of E. coli; and the EPM of Cytophaga U67 and Acholeplasma laidlawii remained unaltered by CCCP when measured in their respective growth media. Extrusion of filamentous bacteriophage f1 from cells of its host, E. coli A95, caused a shift to a higher negative EPM. We also measured a variety of Gram-positive strains, all of which displayed different EPMs. When membrane fractions of E. coli were adsorbed to latex spheres, characteristic differences between the EPM of beads coated with either inner or outer membrane were observed. The results suggest that the rapid EPM analysis is a useful tool to study the net electric charge of microorganisms and to examine changes of surface properties during interaction of cells with viruses, proteins (antibody) and charged antibiotics.     

Surface Charge of Trypanosoma cruzi. Binding of Cationized Ferritin and Measurement of Cellular Electrophoretic Mobility*

SYNOPSIS The surface charge of epimastigote and trypomastigote forms of Trypanosoma cruzi was evaluated by means of binding of cationized ferritin to the cell surface as visualized by electron microscopy, and by direct measurements of the cellular microelectrophoretic mobility (EPM). Epimastigote forms had a mean EPM of -0.52 μm^.s^-1.V^-1.cm and were lightly labeled with cationized ferritin. In contrast, bloodstream trypomastigotes had a much higher EPM (-1.14), and the surface was heavily labeled with cationized ferritin. When trypomastigotes from stationary phase cultures were isolated on DEAE cellulose columns, the mean EPM was found to be significantly lower (-0.63), and labeling with cationized ferritin decreased. With a mixed population containing epimastigote, trypomastigote, and intermediate forms, EPM values ranging between -0.70 to -1.14 were found. From these observations we conclude that there is a definite increase in negative surface charge during development from epi- to trypomastigote forms of T. cruzi.     

Phytohemagglutinin-induced change in the distribution of acidic sugars in surface membrane of lymphoid cells and blocking of the radiation effect.

Cell electrophoretic mobilities (EPM) of cultured lymphoblastoid cells were measured after removal of acidic sugars to investigate whether the localization of these acidic sugars was altered by the action of phytohaemagglutinin (PHA). After treatment with neuraminidase or hyaluronidase, the EPM of control cells decreased 50.1 and 0.3%, while that of PHA-treated cells decreased 25.2 and 39.0%, respectively. These results suggest that hyaluronic acid appeared at the periphery of the cell surface in place of some sialic acid after incubation with PHA. The change became evident after 10 min incubation with PHA and reached its maximum after 20 min at 37 °C, but no change was observed at 4 °C. The EPM decreased with time after X-irradiation, and reached a minimum value after 4 h. The addition of PHA to culture before irradiation completely blocked the X-ray-mediated reduction in EPM. PHA administration after irradiation stopped further EPM reduction. These results seem to suggest a rapid rearrangement of membrane molecules linking with the receptors and acidic sugars induced by PHA, and blocking of further conformation change by X-irradiation.     

Charge density on native and ultraviolet-irradiated myosin A

The axial ratios (a/b) of native and 60 min. ultraviolet-irradiated myosin A molecules were calculated from previously reported sedimentation and diffusion data; values found were a/b = 74, a = 1880 A., and b = 26 A. for native myosin A; a/b = 104, a = 3280 A., and b = 32 A. for 60 min. ultraviolet-irradiated myosin A. Electrophoretic mobilities gave identical values of 3.2 (±0.1) × 10^?5 cm.²/v.-sec. for both native and 60 min. ultraviolet-irradiated myosin A. From the prior sedimentation and diffusion data, together with newly obtained electrophoretic data, the net charge Z and the charge density σ of native and ultraviolet-irradiated myosin A molecules were calculated from Henry's equation. The following results were obtained: for native myosin A, Z = 160 negative charges per molecule and σ = 22 coulombs/cm.²; for ultraviolet-irradiated myosin A, Z = 312 negative charges per molecule and σ = 20 coulombs/cm.². The results of this study provide an experimental demonstration that, the electrophoretic mobility of charged solute particles is dependent upon the particle charge density and not on the absolute charge of the particle.     

Microwaves (2,450 MHz) suppress murine natural killer cell activity

The effect of 2,450-MHz CW microwaves on natural killer (NK) cell activity and lymphocyte responsiveness to mitogen stimulation was studied in mice. Groups of mice were irradiated at power densities of 5, 15, or 30 mW/cm2 (SAR = 3.5, 10.5, and 21 W/kg respectively) for 1.5 h on 2 or 9 consecutive days. NK cell activity was determined using an in vitro 51Cr release cytotoxicity assay and an in vivo tumor-cell clearance assay. No consistent change was observed in the mitogen response of spleen cells from sham compared with irradiated mice. A significant suppression of NK cell activity measured in vitro was observed for mice irradiated at 30 mW/cm2, but not at 15 or 5 mW/cm2. A significant suppression of NK cell activity, as determined using the in vivo tumor clearance assay, was also observed at 30 mW/cm2. NK cell activity, as determined using the in vitro assay, returned to normal within 24 h following the last irradiation. Treatment of mice with hydrocortisone caused suppression of NK cell activity measured in vitro and in vivo. Paradoxically, peritoneal macrophage phagocytosis was enhanced following irradiation at 30 mW/cm2, the power density at which NK activity was suppressed. The possible role that microwave heating plays in producing these effects is discussed.     

Dissociation of high density lipoprotein precursors from apolipoprotein B-containing lipoproteins in the presence of unesterified fatty acids and a source of apolipoprotein A-I

Incubation of low (LDL), intermediate (IDL), or very low density lipoproteins (VLDL) with palmitic acid and either high density lipoproteins (HDL), delipidated HDL, or purified apolipoprotein (apo) A-I resulted in the formation of lipoprotein particles with discoidal structure and mean particle diameters ranging from 146 to 254 A by electron microscopy. Discs produced from IDL or LDL averaged 26% protein, 42% phospholipid, 5% cholesteryl esters, 24% free cholesterol, and 3% triglycerides; preparations derived from VLDL contained up to 21% triglycerides. ApoA-I was the predominant protein present, with smaller amounts of apoA-II. Crosslinking studies of discs derived from LDL or IDL indicated the presence of four apoA-I molecules per particle, while those derived from large VLDL varied more in size and contained as many as six apoA-I molecules per particle. Incubation of discs derived from IDL or LDL with purified lecithin:cholesterol acyltransferase (LCAT), albumin, and a source of free cholesterol produced core-containing particles with size and composition similar to HDL2b. VLDL-derived discs behaved similarly, although the HDL products were somewhat larger and more variable in size. When discs were incubated with plasma d greater than 1.21 g/ml fraction rather than LCAT, core-containing particles in the size range of normal HDL2a and HDL3a were also produced. A variety of other purified free fatty acids were shown to promote disc formation. In addition, some mono and polyunsaturated fatty acids facilitated the formation of smaller, spherical particles in the size range of HDL3c. Both discoidal and small spherical apoA-I-containing lipoproteins were generated when native VLDL was incubated with lipoprotein lipase in the presence of delipidated HDL. We conclude that lipolysis product-mediated dissociation of lipid-apoA-I complexes from VLDL, IDL, or LDL may be a mechanism for formation of HDL subclasses during lipolysis, and that the availability of different lipids may influence the type of HDL-precursors formed by this mechanism.     

Increased trypsin sensitivity of cell surface macromolecules after malignant transformation.

The effect of treatment with 0.04% (w/v) trypsin (EC 3.4.4.4) for 3 h on the electrophoretic mobility (EPM) of polyoma-virus malignantly transformed BHK21 cells (Py6) and their normal counterparts has been investigated. These particular conditions were chosen because an earlier study had shown that such treatment released material from the Py6 cells which was not obtained from the BHK21 cells. The negative EPM of the Py6 cells at pH 7.5 was greatly increased by this treatment; whereas the EPM of the BHK21 cells remained unchanged. Active enzyme was required to produce the change. No evidence was obtained for cytolysis, cytotoxicity or uptake of the enzyme by the treated Py6 cells. Measurement of the EPM of the Py6 cells at different pH levels before and after trypsin treatment suggested that the enzyme was removing cationic groupings from the cell surface.     

Partial delipidation improves the T-cell antigenicity of hepatitis B virus surface antigen

Hepatitis B virus surface antigen (HBsAg) is a complex macromolecular particle composed of glycoproteins and lipids. The latter, representing 25% of the particle mass, are of host origin and determine the solubility, stability, and, indirectly, B-cell immunogenicity of HBsAg. HBsAg is a T-cell-dependent immunogen that does not elicit a detectable humoral immune response in 5% of HBsAg vaccine recipients and in most subjects suffering from chronic hepatitis B. We investigated the influence of the lipid content on the antigenicity of the particle. Lipids were partially removed from HBsAg by treatment with beta-D-octyl glucoside and density centrifugation. Sham treatment consisted of density centrifugation of HBsAg only. We compared the in vitro proliferative responses of established T-cell lines and nonfractionated peripheral blood mononuclear cells (PBMC) from HBsAg vaccinees and chronic HBV patients when stimulated with partially delipidated HBsAg, untreated HBsAg, or sham-treated HBsAg. In all experiments, delipidated HBsAg turned out to be 10 to 100 times more antigenic than its untreated or sham-treated counterpart. Remarkably, PBMC from vaccine nonresponders or chronic HBV patients displayed a proliferative response towards delipidated HBsAg, whereas native HBsAg never induced a response. A series of control experiments demonstrated that this enhancement of T-cell antigenicity was HBsAg specific and directly linked to lipid extraction. Nonspecific adjuvant effects of any kind could be ruled out. In vivo evaluation in mice demonstrated that delipidated particles lose most of their B-cell antigenicity. However, when native and delipidated particles were mixed, these mixtures induced equal or slightly superior anti-HBs responses to those induced by the same quantity of native HBsAg alone. In conclusion, our data show that partial delipidation of HBsAg strikingly increases the T-cell antigenicity of this unique viral antigen.     

A sensitive capillary GC assay for the determination of sparteine oxidation products in microsomal fractions of human liver

A sensitive method for the assay of sparteine oxidase activity in vitro by microsomal fractions of human liver is described. The activity of sparteine oxidase was assessed by the formation of 2- and 5-dehydrosparteines, which were estimated by capillary gas chromatography with N2-FID detection. The limit of detection of the two metabolites, 2- and 5-dehydrosparteine, was 10 pmol (2.3 ng) per sample. Sparteine oxidase activity was linear with microsomal protein concentration ranging from 25 to 200 ug and with incubation times between 5 and 60 minutes. Omission of NADPH on incubation under an atmosphere of carbon monoxide inhibited formation of both metabolites, thus indicating that aforementioned metabolites arise in reaction catalyzed by cytochrome P-450. In three liver samples from humans classified as extensive (EM) metabolizers the formation of 2- and 5-dehydrosparteines was observed, 2-dehydrosparteine being the major metabolite. In these samples sparteine oxidase activity was characterised by Vmax = 136 +/- 53 pmol/min/mg and Km = 44 +/- 12 microM for 2-dehydrosparteine formation. For 5-dehydrosparteine formation the following values were obtained: Vmax = 57 +/- 18 pmol/min/mg and Km = 42 +/- 26 microM. In a liver sample from a poor metabolizer (PM) only the formation of 2-dehydrosparteine was detected with the method of analysis used. In this sample a great increase in Km (Km PM = 3033 microM) was noted, while Vmax was very similar to those obtained for 2-dehydrosparteine formation in EM subjects (Vmax PM = 147 pmol min/mg).     

Acceleration of oxidative folding of bovine pancreatic ribonuclease A by anion-induced stabilization and formation of structured native-like intermediates

Phosphate anions accelerate the oxidative folding of reduced bovine pancreatic ribonuclease A with dithiothreitol at several temperatures and ionic strengths. The addition of 400 mM phosphate at pH 8.1 increased the regeneration rate of native protein 2.5-fold at 15 degrees C, 3.5-fold at 25 degrees C, and 20-fold at 37 degrees C, compared to the rate in the absence of phosphate. In addition, the effects of other ions on the oxidative folding of RNase A were examined. Fluoride was found to accelerate the formation of native protein under the same oxidizing conditions. In contrast, cations of high charge density or ions with low charge density appear to have an opposite effect on the folding of RNase A. The catalysis of oxidative folding results largely from an anion-dependent stabilization and formation of tertiary structure in productive disulfide intermediates (des-species). Phosphate and fluoride also accelerate the initial equilibration of unstructured disulfide ensembles, presumably due to non-specific electrostatic and hydrogen bonding effects on the protein and solvent.     

Response of the headgroup of phosphatidylglycerol to membrane surface charge as studied by deuterium and phosphorus-31 nuclear magnetic resonance

The response to membrane surface charge of the glycerol headgroup of dimyristoyl-phosphatidylglycerol (DMPG) was investigated via deuterium and phosphorus-31 nuclear magnetic resonance spectroscopy. The membrane surface charge was manipulated by adding various amounts of neutral dimyristoylphosphatidylcholine (DMPC) and/or positively charged didodecyldimethylammonium bromide (DDAB) to the negatively charged DMPG, selectively deuterated at the alpha and beta segments of its glycerol headgroup. The deuterium and phosphorus-31 nuclear magnetic resonance spectra were all characteristic of random dispersions of liquid-crystalline lipids in a bilayer configuration. Differential scanning calorimetry showed that all mixtures investigated exhibited gel to liquid-crystalline phase transitions below 35 degrees C. Measurements of the deuterium quadrupole splitting and of the phosphorus-31 chemical shift anisotropy lead to the following observations. (1) Dilution of the negative surface charge density by the addition of DMPC had little effect on the quadrupole splitting from either alpha- or beta-deuterated DMPG. (2) Direct cancellation of the negative surface charge density by addition of DDAB led to a progressive decrease in the quadrupole splitting measured from alpha-deuterated DMPG, while the quadrupole splitting measured from beta-deuterated DMPG increased. For alpha-deuterated DMPG addition of 0.3 mole fraction of DDAB resulted in the appearance of two distinct quadrupole splittings. No such effect was observed for beta-deuterated DMPG.     

Recovery of surface membrane in anterior pituitary cells. Variations in traffic detected with anionic and cationic ferritin

Cells dissociated from rat anterior pituitaries were incubated with native or cationized ferritin (CF) to trace the fate of surface membrane. Native ferritin, which did not bind to the cell surface, was taken up in small amounts by bulk-phase endocytosis and was found increasingly (over 1-2 h) concentrated in lysosomes. CF at 100-fold less concentrations bound rapidly to the cell membrane, was taken up by endocytosis in far greater amounts, and within 15-60 min was found increasingly within multiple stacked Golgi cisternae, around forming secretion granules, and within elements of GERL, as well as within lysosomes. The findings demonstrate that the fate of the tracer--and presumably also that of the surface membrane--varies with the same molecule differing only in net charge: vesicles carrying anionic ferritin (net negative charge) fuse only with elements of the lysosomal system whereas those carrying CF (net positive charge) can fuse not only with elements of the lysosomal system, but also with elements along the secretory pathway (Golgi cisternae and condensing granules) as well.     

ZETA-Potential and surface charge of Thermoplasma acidophila.

The surface charge density and the zeta-potential of Thermoplasma acidophila was estimated from microscopic electrophoresis experiments. The cells moved towards the positive electrode. The mobility remained constant from pH 2 to 5, and increased for pH values higher than 6. The mobility at pH 6 decreased dramatically with increased external Ca2+ concentration. At pH 2 and an ionic strength similar to that of the growth medium, the zeta-potential was about 8 mV, negative relative to the bulk medium; the surface charge density was 1360esu/cm-2 which corresponds to one elementary charge per 3500 A2.