Decrease In Electrophoretic Mobility Research Articles

Electrophoretic Properties of Cowpea Mosaic Virus (Severe Subgroup)

SUMMARY Particles of the Puerto Rico isolate of cowpea mosaic virus (CPMV-PR), which belongs in the severe subgroup, had one electrophoretic form when analysed by 2·5% polyacrylamide gel electrophoresis and zone electrophoresis in sucrose gradients at pH 7·8, 7·5, and 5·5. The electrophoretic properties of CPMV-PR did not vary with time after inoculation but the virus could be converted to a slower migrating form by in vitro treatment with trypsin. The partial conversion by trypsin was accompanied by and was possibly the result of proteolytic conversion of the S protein subunit to a lower molecular mass form. Analysis of proteins of untreated CPMV-PR suggested that the S subunit of the virus could also be converted in vivo by plant proteases to a smaller form without any change in the particle net surface charge, while in vitro treatment with trypsin affected those S subunits not previously converted by plant proteases, by converting them to a form slightly larger and with a greater positive charge than the S subunit produced by action of the plant proteases. This hypothesis accounts both for the decrease in electrophoretic mobility and for the fact that the conversion was only partial.

Mechanisms by which ultrasound stimulates tissue repair

Experimental studies of tissue repair in animals have shown that therapeutic levels of ultrasound can increase the rate of growth of replacement tissue at sites of injury. In a controlled clinical trial, similar results were obtained when chronic varicose ulcers were treated with similar levels of ultrasound. Although local heating may be partly involved in the stimulation of repair by ultrasound, it is not the sole cause of it. Nonthermal mechanisms proposed include acoustic streaming, possibly associated with cavitation. The irradiation of fibroblasts with therapeutic levels of ultrasound at ambient pressure resulted in (a) stimulation of collagen synthesis, (b) decrease in electrophoretic mobility, (c) modification of locomotor activity, (d) temporary elevation of intracellular calcium, (e) ultrastructural changes including plasma membrane alteration and increased vacuolation. There was no change in the viability of the fibroblasts. All the changes found were suppressed in matched cell samples irradiated in the same manner at elevated pressure, suggesting the involvement of cavitation. The cellular effects described are consistent with those expected to be associated with the enhancement of tissue repair. [Work supported by SRC and Guy's Arthritis Research Unit.]

Affinity electrophoresis of proteins interacting with blue dextran

Interaction of several enzymes (pyruvate kinase, myokinase, creatine kinase, aldolase, malate dehydrogenase, lactate dehydrogenase, alcohol dehydrogenase and glucose-6-phosphate dehydrogenase) and other proteins (bovine serum albumin and ovalbumin) with Blue Dextran was studied by means of affinity electrophoresis in polyacrylamide gels. A decrease of electrophoretic mobility of enzymes in affinity gels was dependent on Blue Dextran concentration and in some cases, dissociation constants of the protein-immobilized dye complexes could be calculated. Affinity electrophoresis in the presence of Blue Dextran reveals in some cases additional bands of isoenzymes, as compared with the control gels (without Blue Dextran).

Electrophoretic Mobility Distributions of Normal Human T and B Lymphocytes and of Peripheral Blood Lymphoblasts in Acute Lymphocytic Leukemia: Effects of Neuraminidase and of Solvent Ionic Strength

Human T and B lymphocytes were found to be distinguishable on the basis of electrophoretic mobility, with the T cells having the higher mobility, in agreement with previous reports. The effects of the enzyme neuraminidase on the electrophoretic mobilities of T and B lymphocytes were determined. T lymphocytes showed a greater decrease in electrophoretic mobility after neuraminidase treatment; the relative mobilities of T and B cells were reversed by neuraminidase treatment, and the electrophoretic distinguishability was enhanced. The electrophoretic mobility distributions of lymphoblasts from patients with acute lymphocytic leukemia were found to differ from those of normal cells in their response to neuraminidase treatment and to changes in solution ionic strength. These results imply that the surface structure of the leukemic cells differs from that of either T or B lymphocytes from normal donors.

Effect of erythrocytic chalone on electrophoretic mobility of mouse bone marrow cells

Five fractions were isolated by cell electrophoresis in a Ficoll density gradient from the bone marrow of mice receiving physiological saline (control) and erythrocytic chalone (experiment). The absolute and relative distribution of the cells in the control and experimental fractions differed: the maximal number of cells in the control (up to 78%) was found in fractions 3–5 and the minimal (up to 22%) in fractions 1–2. A redistribution of the cellular material in all the fractions took place 25 min after treatment with erythrocytic chalone. The number of cells in fraction 1 was particularly increased (by 2.6 times). Analysis of the “myelograms” of films from fraction 1 in the experimental and control series showed that the number of cells in fraction 1 was increased on account of cells of the erythroid series. Among the proliferating cells of the erythron, the greatest decrease in electrophoretic mobility was observed in the proerythroblasts. It is suggested that as a result of interaction between erythrocytic chalone and membrane receptors of proliferating cells of the erythron their electric charge is reduced and changes take place in intracellular processes leading to delay in mitosis.

LYMPHOCYTE SENSITIZATION IN CHILDREN WITH LYMPHOBLASTIC LEUKEMIA AND LYMPKOMA AS MEASURED BY THE ELECTROPHORETIC MOBILITY TEST

A modified electrophoretic mobility (EM) test was performed in 53 children with lymphoblastic leukemia (ALL) and lymphoma to examine their lymphocyte sensitization to myelin basic protein (encephalitogenic factor). Measurements in the cytopherometer were facilitated by using devitalized sheep erythrocytes as indicator particles instead of macrophages.A significant decrease in electrophoretic mobility as compared to an 0-standard was found in 36 of 41 patients with ALL and in 12 of 12 patients with lymphoma; thus giving a sensitivity rate of 88 to 100 %. Only 1 out of 10 healthy individuals and 34 of 38 children with non-malignant disorders (autoimmune diseases excluded) also had a positive EM-test. Almost all patients with ALL were in hematological remission either on or off therapy. 4 of the 5 non-reactive children with ALL were at diagnosis or in phase I of induction therapy. No striking change in lymphocyte reactivity was seen between lymphoblastic leukemia, Hodgkin's or Non-Hodgkin's lymphoma.These results indicate that patients with lymphoid malignancies have remaining lymphocytes which had been sensitized by a common antigen of the malignant cell clone in the beginning of the disease.

Lactate dehydrogenase in amoeba, Acanthamoeba sp. in different phases and conditions of culture

1. 1. The activity and kinetic changes of amoeba LDH in different phases and conditions of culture were investigated. 2. 2. LDH of the amoeba is specific against d(−)LDH irrespective of the hypoxic conditions created. 3. 3. In hypoxic conditions it was not possible to visualize the presence of another LDH isozyme of muscle type by kinetic or electrophoretic analysis. 4. 4. However, the changes in the K m value and the L:H ratio as well as the decrease of electrophoretic mobility of LDH band indicate the change in kinetic properties of the enzyme from an obviously heart type in oxygenated culture in the direction of a muscle type LDH in strongly hypoxic culture conditions. 5. 5. The influence of factors producing either environmental or metabolic hypoxia on possible repression or induction of LDH in amoeba is discussed.

Lymphocyte sensitization in childhood solid tumours and lymphoblastic leukaemia, measured by electrophoretic mobility test.

A modified electrophoretic mobility (EM) test was performed in 150 children to examine their lymphocyte sensitization to myelin basic protein (encephalitogenic factor). Measurements in the cytopherometer were facilitated by using devitalized sheep erythrocytes as indicator particles instead of macrophages. A significant decrease in EM was found in 29/30 children with acute lymphoblastic leukaemia and in 67/75 children with solid tumours, thus giving a false negative rate in malignant disease of 9/105=8-6%, as compared to 6 false positives among 45 children with non-malignant disorders; 5 of the later "false/positive" 6 patients had autoimmune disease. Results of the EM test in the children with leukaemia were compared with those in 9 patients with non-Hodgkin's lymphoma and 2 with Hodgkin's disease at different stages, but no striking change was seen between different diseases, or after cessation of long-term immunosuppressive chemotherapy. Percentage of "slowing" ranged from 4 to 30%. These results indicate that patients with lymphoid malignancies still have lymphocytes which had been sensitized by a common antigen of the malignant cell clone at the beginning of the disease. The EM test, furthermore, could serve as an additional diagnostic aid in differentiating benign from malignant masses in the abdomen, extremities or intracranial disease.

Characterization of polyoma virus T antigen.

High-titer antiserum raised in rats against the tumor (T) antigen of polyoma virus was used to purify the T antigen by the Staphylococcus protein A antibody adsorbent technique. Sodium dodecyl sulfate/polyacrylamide gel electrophoresis allowed the identification of a protein with an apparent molecular weight of 100,000-108,000 as a major component induced in lytically infected mouse cells. In cells infected by ts A mutants this component was temperature sensitive. Several minor components were also observed. In pulse and chase experiments there was a slight decrease in electrophoretic mobility of T antigen during the chase period at the permissive temperature, suggesting that the T antigen is a modified protein. In two lines of transformed cells, the amount of T antigen seemed to be considerably less than in lytically infected cells, but the size of the antigen appeared to be equal.

Target of X Irradiation and Dislocation of Sialic Acid in Decrease of Cell Surface Charge of Erythrocytes

SATO, C., KOJIMA, K., AND NISHIZAWA, K. Target of X Irradiation and Dislocation of Sialic Acid in Decrease of Cell Surface Charge of Erythrocytes. Radiat. Res. 69, 367-374 (1977). Rat erythrocytes and their ghosts were utilized to investigate the target and mechanism of the reported decrease in electrophoretic mobility (EPM) of cultured mammalian cells after exposure to X radiation. Similar dose-dependent reductions in EPM's were observed in both whole erythrocytes and their ghosts. Recovery of EPM was evident in whole erythrocytes after irradiation with doses lower than 500 R, but was lacking in ghosts. This suggests that the cell membrane itself is the target of X irradiation with respect to EPM reduction, and that a cytoplasmic factor is necessary for the recovery. The amount of sialic acid did not decrease after irradiation. By using phosphate buffers of different ionic strengths, which determines the effective thickness of the ion atmosphere for EPM, dislocation of sialic acid from the peripheral zone of 0-7.5 'A to a deeper zone of 9.7-17 A' was suggested. The decrease in EPM was independent of temperature in the range 22-370C, but was markedly influenced by temperature between 10 and 200C. This temperature dependence of EPM was similar to that of the fluidity of membrane lipid, which determines the facility in the rearrangement of membrane components.

Concanavalin A-Induced Decrease in Cell Surface Charge and its Modification by Sulfhydryl Blocking Agents

The electric surface charge of mouse melanoma B 16-C 2 W cells treated with various concentrations of Con A was determined by cell electrophoresis. At Con A concentrations higher than 5 μg/ml, the cellular electrophoretic mobility decreased significantly at 10 minutes incubation, but this effect was reversed by α-methyl-D-mannoside. At these higher Con A concentrations, more than 30% of Con A receptors were bound with lecitn. Pretreatment of cells with sulfhydryl blocking agents, 10-5 M p-chloromercuribenzoic acid or 10-5M N-ethylmaleimide, completely prevented the subsequent Con A-induced mobility reduction without changing the amount of Con A-binding to the cell surface. Similar effects were found with pretreatment with protein linkage agents, such as 2% glutaraldehyde and 10-6M fluorescein mercuric acetate. The removal of chondroitin sulfate with the specific enzyme resulted in a 30% decrease in electrophoretic mobility of intact cells but produced no change in Con A-treated cells. These results suggest that Con A treatment may induce an alteration in chondroitin sulfate distributions at the cell surface and that sulfhydryl residues of membrane proteins may play an important role in this surface alteration.

Effect of incubation in human plasma on electrophoretic mobility of brain-type creatine phosphokinase

1. 1. Electrophoretic mobility of brain-type creatine phosphokinase (CPK) from rabbit, rat and human decreased after incubation in human plasma for 3 h or 17 h but did not change after incubation in isotonic saline solution. The presence of the reducing agent mercaptoethanol during incubation in human plasma tended to reduce the rate of change of electrophoretic mobility of BB-CPK. 2. 2. The activity of brain CPK was almost completely lost after incubation for 17 h in human plasma. The addition of mercaptoethanol after incubation of brain CPK in human plasma could only partially restore the enzyme activity. 3. 3. The decrease in electrophoretic mobility of rat brain CPK occurred after incubation in an ultra-filtrate of human plasma which contained molecules whose molecular weight did not exceed 1000. 4. 4. Gel chromatography indicated that incubation of brain CPK in human plasma did not markedly change the molecular size of the enzyme.

The detection of phytomitogen-induced changes in human lymphocyte surfaces by laser doppler spectroscopy

The changes in electrophoretic mobility and isoelectric point produced by incubating human peripheral blood lymphocytes with phytohemagglutinin (PHA) and concanavalin A (Con A) have been characterized by laser Doppler spectroscopy. The results extend and partially confirm older observations made by classical procedures. Incubation with either agent for 90 min at 37°C resulted in stable and reproducible decreases in electrophoretic mobility, and increases in the isoelectric point. The incubation conditions used are known to permit primary attachment of the phytomitogen, capping and endocytosis nevertheless, at least in the case of Con A, washing the cells with a specific inhibitor for Con A binding, methyl-α-D-glucoside (MAG), resulted in complete reversal of the electrokinetic changes, showing that the underlying changes in cell surface constitution detected under these conditions are solely due to reversibly bound Con A. The results suggest that laser Doppler spectroscopic changes could provide a direct assay for specific binding to immunocompetent cell surfaces.

Cell surface charge and regulation of cell division of 3T3 cells and transformed derivatives

The cell surface charge of 3T3, 3T6, SV40-3T3 cells and trypsin-, neuraminidase- and serumtreated preparations of these has been characterized by microcell electrophoresis. At 25 °C, density-inhibited 3T3 cells show a decrease in electrophoretic mobility when treated with various stimuli of cell division. This effect is not observed at 25 °C for transformed derivatives. The surface charge configuration of various cell preparations exhibits a thermal transition which is located within a temperature range characteristic of each preparation. These and other results from cell electrophoresis, taken together with those obtained in agglutination studies by other authors, are considered evidence for the occurrence in the plasma membrane of these cells of a twodimensional phase separation. The temperature range of this phase separation is shifted on treating the cells by growth stimuli. This effect might be an indication of a basic trigger mechanism in the cell cycle.

Role of sialic acid in erythrocyte survival

The role of membrane sialic acid in erythrocyte survival is unclear, although there is evidence for a reduction in sialic acid and surface charge in older erythrocytes. We reduced the surface charge of human, rat, and rabbit erythrocytes by removing sialic acid with neuraminidase.Reduction in sialic acid correlated with decreases in electrophoretic mobility and loss of PAS staining of membrane glycoproteins on polyacrylamide gels. No changes in ATP levels or deformability were found. 51Cr erythrocyte survivals in rats and rabbits showed rapid clearance of desialylated erythrocytes with sequestration by the liver.These results suggest that reduction in erythrocyte sialic acid is a mechanism of erythrocyte destruction and may be important in erythrocyte senescence.

Role of Sialic Acid in Erythrocyte Survival

The role of membrane sialic acid in erythrocyte survival is unclear, although there is evidence for a reduction in sialic acid and surface charge in older erythrocytes. We reduced the surface charge of human, rat, and rabbit erythrocytes by removing sialic acid with neuraminidase. Reduction in sialic acid correlated with decreases in electrophoretic mobility and loss of PAS staining of membrane glycoproteins on polyacrylamide gels. No changes in ATP levels or deformability were found. 51Cr erythrocyte survivals in rats and rabbits showed rapid clearance of desialylated erythrocytes with sequestration by the liver. These results suggest that reduction in erythrocyte sialic acid is a mechanism of erythrocyte destruction and may be important in erythrocyte senescence.

Cytochalasin B and the sialic acids of Ehrlich ascites cells

The effect of cytochalasin B (CB) on the electrophoretic mobility and density of ionized sialic acid groups at the surface of Ehrlich ascites cells was examined together with a biochemical assay of the total sialic acid content of treated and control cells. Sialic acid assays indicated that CB-treated cells had a greater amount of total sialic acid and sialic acid sensitive to neuraminidase than control cells/cell. Equal amounts of sialic acid were removable by neuraminidase treatment from control cells and cells pretreated with neuraminidase and subsequently cultured with CB. The electrophoresis results showed a decrease in electrophoretic mobility in the presence of CB which could be reversed by growth in CB-free medium. Neuraminidase treatment did not make a significant additional reduction in the mobility of CB-treated cells. CB also prevented the recovery of electrophoretic mobility of neuraminidase treated cells. The results suggest that while CB does not inhibit sialic acid synthesis, it does alter the expression of ionized sialic acid groups at the electrokinetic surface. CB-containing culture media could be re-utilized several times suggesting that CB is not significantly bound or metabolized by Ehrlich ascites cells.

A study of the cell surface of tumour, foetal and lymph-node cells by cell electrophoresis after antibody and enzymic treatment

1. Rabbit anti-(rat foetal liver) serum, absorbed with adult rat liver cells, decreased the electrophoretic mobility of foetal liver cells by 51% and rat hepatoma cells by 45%, indicating the presence of a foetal-type antigen on the hepatoma cell membrane. 2. The chemical nature of the surface antigen was investigated. Incubation with neuraminidase had no effect on adult liver cells but decreased the electrophoretic mobility of foetal liver cells by 51% and of hepatoma cells by 34%; the effect of antiserum was decreased to one-fifth. 3. Sialic acid, or the supernatant from neuraminidase-treated cells, partially blocked the decrease in electrophoretic mobility induced by antiserum. 4. The pH-electrophoretic mobility curves of hepatoma cells treated with antisera were consistent with a sialic acidcontaining antigen on the surface of the tumour cells. 5. Treatment with ribonuclease did not decrease the electrophoretic mobility of adult-liver cells, but decreased that of the foetal liver cells by 17% and hepatoma cells by 29%. 6. In parallel studies made with mouse BP8 ascites-tumour cells ribonuclease decreased the electrophoretic mobility by 39%, that of normal mouse lymph-node cells by 4.8% and allergized mouse lymph-node cells by 13.3%. 7. Trypsin treatment also decreased the electrophoretic mobility of hepatoma cells by 22%.

Study of soluble lipoprotein in rat liver mitochondria.

1. A water-soluble lipoprotein was isolated and purified from osmotically shocked preparations of rat liver mitochondria by using a technique of Sephadex-sandwich disc electrophoresis. 2. The purified lipoprotein migrates as a distinct sharp zone in high-resolution electrophoretic systems, indicating high degree of purity. 3. The lipoprotein resembles mitochondrial membranes with respect to lipid composition and lipid/protein ratio. 4. The lipoprotein and its apoprotein fraction obtained by delipidization at -18 degrees C to -20 degrees C have common properties with respect to their fluorescence spectra, instability to storage and electrophoretic mobility. 5. The purified lipoprotein has an excitation maximum at 325nm and a fluorescence maximum at 418nm. 6. Storage at 4 degrees C for 4 days or repeated freezing and thawing results in 15-30% decrease in electrophoretic mobility. 7. The patterns of incorporation in vitro of [1-(14)C]leucine into proteins of the soluble lipoprotein and of mitochondrial membrane of isolated rat liver mitochondria suggest a probable precursor role for the apoprotein in the formation of mitochondrial membrane protein. 8. Lipoprotein preparations isolated from mitochondrial fractions of rat kidney, brain and heart and of chicken and mouse liver resemble closely that obtained from rat liver mitochondria, suggesting that the soluble lipoprotein could be a distinct entity of mitochondrial origin.

The contribution of sialic acids to the surface charge of human tooth particles

The amounts of sialic acids in tooth particles were assayed by chemical methods, and the contribution of these ionized moieties to the surface charge of the particles were studied by electrokinetic methods. Using chemical analysis, sialic acid was demonstrated in dentine and cementum, but not in enamel. The reduction in electrophoretic mobility following incubation with low concentrations ( 5 units 12 ml ) of active neuraminidase was used as evidence for the presence of sialic acid moieties at the electrokinetic surface of cementum and enamel particles. Dentine particles could not be analyzed as their electrophoretic mobilities were too low to yield reliable data. It was also demonstrated that active and inactive neuraminidase in high concentrations ( 250 units 12 ml ) readily adsorbed to enamel, cementum, calculus and hydroxyapatite, as shown by the decrease of electrophoretic mobility of these particles.