Mean Electrophoretic Mobility Research Articles

Alteration by prolactin of surface charge and membrane fluidity of rat 13762 mammary ascites tumor cells.

Intraperitoneal injection of ovine prolactin (100 micrograms/d) in Fischer 344 rats bearing transplantable 13762 mammary ascites tumor (MAT) cells modifies the surface charge density and membrane fluidity of the tumor cells. In each of five experiments the mean electrophoretic mobility (epm) of MAT cells taken from prolactin-treated rats was significantly lower than that of cells from nonhormone-treated controls. Prolactin concentrations were increased in vivo by (a) direct intraperitoneal injection of ovine prolactin; (b) subcutaneous implantation of diethylstilbestrol-containing silastic capsules to produce pituitary prolactin secreting tumors; or (c) a single subcutaneous injection of polyestradiol phosphate, a long-acting estrogen. In an effort to establish that the prolactin effect was a direct one, two in vivo protocols were used: (a) MAT cells were coincubated with anterior pituitary halves obtained from nontumor-bearing littermates; or (b) rat or ovine prolactin was added to serum-free culture media containing MAT cells. In both protocols, the epm of the prolactin-treated cells was significantly lower. The isoelectric focusing pH of whole cells was increased by prolactin treatment from 4.93 to 5.12, consistent with a reduction in the number of surface carboxyl groups. The fluidity of membranes of treated cells was drastically increased, as measured by spin-label probe rotation rates. These combined results imply that the hormone exerts its effect by stimulating events in the cell that lead to a reduction of the average density of carboxylic acid residues on the tumor cell surface.

Factors in the sera of tumor-bearing mice modifying the charge of immune cells.

The mean electrophoretic mobility (EPM) of normal C3H spleen cells is significantly slackened (by approximately 15%) when the cells are incubated for 0.5 h at 37 degrees C with serum from slow-growing tumors (VMM2 and VMM1 tumors). On the contrary, it is not modified when the cells are incubated with serum from fast-growing tumors (RV2 or VFM1 tumors). VMM2 serum heated at 56 degrees C induces the same phenomenon. Incubation at 4 degrees C with VMM2 serum does not induce any change in spleen cell EPM. VMM2 and VMM1 sera seem to be essentially active on T cells. Indeed, no effect is observed with cells from 'nude', thymectomized, bone marrow-reconstituted C3H mice, or with the B cell-enriched fraction after nylon wool separation of normal spleen cells. A significant EPM decrease is only observed after serum incubated with the T cell-enriched fraction. Polyacrylamide gel electrophoresis shows that these sera exhibit an increase in the content of different protein groups. These phenomena suggest that the reduction of negative charge of lymphocytes after interaction with some tumor sera (VMM2 and VMM1 sera) might help establish tumor cell-lymphocyte contact and thus explain the slow growth rate of these VMM2 and VMM1 tumors.

Colloid stability of casein-coated polystyrene particles

The stability of casein-coated polystyrene latices with respect to aggregation by sodium chloride has been studied at 20 °C and pH 7.3. Initial aggregation rates have been measured by light scattering as a function of ionic strength (100–1000 mol m–3) for negatively charged particles separately coated with αs1-casein, β-casein, κ-casein and three commercial caseinate mixtures. Casein adsorption reduces the mean electrophoretic mobility of latex particles. At low surface coverage (ca. 10% saturation), derived critical coagulation concentrations are roughly in accord with predictions from DLVO theory based on changes in zeta potential estimated from experimental mobilities. Near saturation coverage, latices are stabilized against aggregation at all ionic strengths up to 1000 mol m–3, presumably by a ‘steric’ mechanism. While each pure casein or caseinate mixture shows the same general behaviour, κ-casein appears quantitatively less effective as a steric stabilizer.

Contact sensitivity to dinitrofluorobenzene and oxazolone in mice: In vitro cell-electrophoretic assay

Prevalent techniques for the assessment of cell-mediated immunity (CMI) in vitro are time consuming and may lack specificity. Deriving from the earlier observation of reduction in mean electrophoretic mobility (EPM) of lymphocytes following the development of humoral immune response, the possibility of using this electrokinetic method for the assessment of contact sensitivity in vitro was tested. CBA mice epicutaneously sensitized to dinitrofluorobenzene and 2-phenyl 4-ethoxy oxazolone showed a marked and reproducible reduction in the mean EPM of their lymph node cells (LNC). The specificity of this alteration in surface charge density was established by the enhancement in the EPM of contact sensitized LNC upon subsequent incubation in vitro with the specific antigen only. The profiles of time kinetics of changes in the EPM of LNC before and after incubation with antigen were virtually parallel to those of in vivo delayed hypersensitivity (DH) reaction as measured by the ear-swelling method. The coefficients of correlation between the reduction in EPM and in vivo DH response for DNFB and oxazolone were 0.89 and 0.86 respectively. EPM measurements may thus provide a sensitive, rapid, and quantitative parameter for the assessment of CMI in vitro.

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 micrometer . s-1 . V-1 . cm. At lower pH, there is a decrease in the negative surface charge with an isoelectric point at pH 1.2. At higher pH (greater than 9.0), there is an increase in the surface charge reaching an EPM of -2.5 micrometers . s-1 . V-1 . cm. 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.

Stimulation and membrane charge changes of spleen cells by factors of low molecular weight associated with murine solid tumors.

Dialysates from a slow-growing VMM2 and a fast-growing RV2 solid murine tumor were fractionated on a Sephadex G-50 column. Similar elution profiles with two peaks were obtained in both cases. Fractions I and II, corresponding to these two peaks were tested for their mitotic properties on lymphocytes in culture and their effect on the negative surface charge of spleen cells by the electrophoretic mobility measurement (EPM) technique. Fraction I from both tumors presents a similar mitogenic action on C3H/He or nu/nu lymphocytes in culture. Incubation for 1 h at 37 degrees C of normal mice spleen cells with fraction I from RV2 or VMM2 tumors did not affect the mean electrophoretic mobility of spleen cells. Fraction II from VMM2 tumor induced a significant decrease in the mean electrophoretic mobility of spleen cells (-13.4%), while fraction II from RV2 tumor increases the EPM (+ 9%) significantly. Incubation with T cell-enriched or B cell-enriched populations or spleen cells from nu/nu mice shows that fraction II from VMM2 interacts with T cells but not with B cells. Fraction II from RV2 increases the T and B cell electrophoretic mobility.

Factors inducing a loss of net negative surface charge on spleen cells of mice grafted with a slow-growing tumour.

The mean electrophoretic mobility (EPM) of splenic cells was determined in 4 different host-tumour systems. In splenic cells harvested from mice bearing slow-growing tumours, a significant EPM decrease was observed (12%) and in increase in the proportion of cells with slow mobility. Moreover, after 1 h incubation in RPMI medium at 37 degrees C and 2 washings, spleen cells showed a marked increase in their EPM (average 30%). Finally, the supernatant from incubation medium after contact with normal spleen cells (1 h at 37 degrees C) produced a significant decrease in their EPM (approximately 12%). On the other hand, no significant EPM variations were found between control spleen cells and cells from fast-growing tumours, before or after 1h incubation. The existence of factors which induce a loss of negative surface charge on spleen cells of some tumour-grafted mice is discussed.

Identification of feline blood B and T lymphocytes by cell electrophoresis

The electrokinetic properties of feline blood lymphocytes isolated by centrifugation over Ficoll-Isopaque gradient were investigated. A biphasic electrophoretic mobility (EPM) distribution was regularly observed with a low-mobility (LM) population (mean EPM: 0.82) accounting for 32% of blood lymphocytes and a high-mobility (HM) population (mean EPM: 1.09) representing 68% of blood lymphocytes. Following fractionation on nylon-wool columns, lymphocytes with B-cell properties (64% sIg +; 9% guinea pig erythrocytes (GPE)-rosette +, PHA and Con A unresponsive) were enriched in the adherent fraction and belonged mainly (78%) to the LM population. In contrast, lymphocytes with T properties (5% sIg +, 42% GPE-rosette +, PHA and Con A responsive) were recovered in the effluent fraction and comprised 84% of HM elements. Thus, in cat blood, LM lymphocytes are likely to represent in majority B cells and HM lymphocytes T cells. This indicates that cell electrophoresis provides an interesting mean for differentiating B and T cells in the cat.

Cotyledonary Storage Proteins in Pisum sativum. III. Patterns of Accumulation During Development

An antiserum raised against proteins from isolated protein bodies of mature pea seeds recognized seven antigenically distinct protein families in cotyledon extracts analysed by crossed immunoelectrophoresis. This antiserum has been used to follow the sequence of appearance and pattern of accumulation of these proteins in peas grown under controlled environmental conditions. One antigen present in mature cotyledons was shown to be a haemagglutinin, one corresponded to legumin, and four distinct, non-crossreacting antigenic species were related to the vicilin series of proteins. Both the number of components detected and the order of their appearance during development were similar in three genotypes examined, but the quantitative representation of legumin relative to other components was genotype-specific. Quantitative differences were observed in the polypeptide composition of the vicilin components responsible for separate antigenic specificities, but qualitative differences were not excluded. During development, similar conspicuous quantitative changes in polypeptide composition, accompanied by a change in mean electrophoretic mobility, occurred amongst the molecules carrying the predominant antigenic determinant related to vicilin (peak 4); again, qualitative differences might also distinguish these components. The antigen responsible for peak 6 contained two major polypeptides distinct from the components of the legumin and vicilin fractions. Peak 6 represented the first fully immunocompetent species to appear during development, but later became quantitatively less significant as the other antigenic species accumuIated. Legumin and the antigen forming peak 6 were first detected immunologically in an apparently incomplete form. These putative precursors disappeared during subsequent development, so that only the corresponding stable precipitin peaks were seen in the immunoelectrophoretic profiles of extracts from mature cotyledons.

Electrokinetic Properties and Mitogen Responsiveness of Mouse Splenic B and T Lymphocytes Following Hydrocortisone Treatment

CBA mice recieved a single intraperitoneal injection of hydrocortisone acetate (OHC) in a dose of 125 mg/kg body weight. At various times therafter, electrophoretic mobility (EPM), surface immunoglobulin (SIG) and in vitro DNA synthetic reactivity to concanavalin A (ConA), phytohemagglutinin (PHA), lipopolysaccharide (LPS) and tuberculin (PPD) were investigated on splenic lymphocytes. OHC was found to deplete rapidly the spleen to a minimum of 18% of control cellularity by day 4 posttreatment. At this time, the proportions of low mobility (LM) and SIG-bearing lymphocytes (B cells) were reduced respectively to 28% (control 54%) and 20% (control 45%). The proportion of high mobility (HM) lymphocytes (T cells) was increased to 72% (control 45%). While the mean EPM of LM cells (0.71) was only slightly and transiently reduced, that of HM cells was significantly augmented (1.24) over control value (1.16). This latter finding was interpreted as indicating the selective removal by OHC of a T cell subpopulation with a mean EPM around 1.10. Changes in mitogenic responsiveness were consistent with these alterations of B and T cell compartments. Despite a marked drop in spontaneous 3H-thymidine uptake, the absolute response to T cell mitogens ConA and PHA remained relatively unchanged. By contrast, the reactivity to B cell mitogens LPS and PPD was strongly depressed. Starting by day 12, regeneration and normalizaiton of lymphocyte populations proceeded slowly and were not achieved before day 26-34.

CELL ELECTROPHORETIC ANALYSIS OF LYMPHOCYTES AND POLYMORPHONUCLEAR CELLS FROM PATIENTS WITH RHEUMATOID ARTHRITIS

Cell electrophoresis was performed on peripheral-blood lymphocytes obtained from 11 patients with rheumatoid arthritis (R.A.) and 16 normal controls. On the basis of cell frequency/mobility histograms each subject's lymphocyte electrophoretic profile was resolved into a bimodal distribution of fast and slow cells. The mean electrophoretic mobility (E.P.M.) of the fast-cell population was similar in controls and in R.A. patients but the mean E.P.M. of the slow population was significantly slower in R.A. than in the controls. In the R.A. patients the E.P.M. of the polymorphonuclear cell population was significantly slower than that of P.M.N. cells in the peripheral blood of controls.

Delineation and Quantitation of Human Peripheral Blood Lymphocyte Subpopulations by Electrophoretic Mobility and Role of Surface Charge in Cell to Cell Interaction

A modified procedure is proposed for determination of electrophoretic mobility (EPM) of lymphocytes which enables readings on 200 cells from a sample size of 5 x 106 lymphocytes. In normal human peripheral blood, B lymphocytes (bearing surface immunoglobulin and receptor for activated C3) were found to carry the lowest charge (0.94 +/- 0.05 mu/sec/V/cm). Their number on the EPM basis was 16.9 +/- 4.2%. Two subpopulations of T lymphocytes, one carrying high charge (TH) with a mean EPM of 1.26 +/- 0.04 mu/sec/V/cm and the other carrying lower charge (TL) with a mean EPM of 1.11 +/- 0.03 mu/sec/V/cm were discernible. The former subpopulation of T cells formed spontaneous rosettes with SRBC in 10 min and the latter represented the remaining T lymphocytes which formed rosettes with 24 hr of incubation with SRBC. The number of early rosette-forming cells increases from 69 to 89% when T cells were incubated with neuraminidase-treated SRBC. These observations suggest that complementary electrostatic charge on T cells and SRBC are among the forces facilitating formation of rosettes between these cells.

Influenza A Virus Interaction with Murine Lymphocytes

Abstract This report describes changes in surface properties of rat thoracic duct lymphocytes (TDL) induced by influenza virus A/Japan 305 (H2N2). After incubation with virus at 37°C, sialic acids were released from the membranes of TDL with concomitant reduction in their mean electrophoretic mobility. Autoradiographs of TDL which had been incubated with 125I virus at 4°C showed that the particles attached to nearly all the lymphocytes although there was variation in the amount of virus bound per cell. Attachment occurred rapidly with maximum labeling evident after incubation at 4°C for 5 min. Incubation at 37°C for 1 hr resulted in a 10 to 18% reduction in the incidence of labeled TDL suggesting that some virus eluted spontaneously. When 125I-virus-treated TDL were incubated in syngeneic serum, substantial amounts of virus eluted reducing the incidence of labeled cells by 50%. In addition, evidence was obtained by indirect immunofluorescence that syngeneic serum contains antibodies which react with sites on the lymphocyte surface exposed by the viral neuraminidase.

Dose-related effect of hydrocortisone treatment on the electrokinetic properties and mitogen responsiveness of mouse thymocytes.

The electrophoretic mobility (EPM) and the ability to synthesize DNA in vitro either spontaneously or in response to concanavalin A (Con A) and phytohemagglutinin (PHA) were investigated on thymocytes from adult female Swiss/B mice which had received intraperitoneally, 2 days previously, various doses (10-750 mg/kg body weight) of hydrocortisone acetate (OHC). A first group of thymocytes, highly sensitive to OHC, rapidly decreased in proportion following administration of increasing doses of OHC (10-125 mg/kg body weight) and included the totality of low-EPM thymocytes (EPM less than 1.0 mum sec-1 V-1 cm) together with about 70% of the high-EPM thymocytes encountered in the normal thymus. The second group of thymocytes (4% of the initial cellularity), which resisted to OHC at 125 mg/kg body weight and was only reduced by half with doses of OHC 6-fold larger, was composed essentially of high-EPM cells. The mean EPM of both groups tended to increase with increasing doses of OHC which suggests that among these two groups thymocytes are all the more OHC-sensitive as they possess a low surface charge. Spontaneous DNA synthesis, measurable on the first 24 h of in vitro cultivation, markedly dropped after treatment with OHC at doses higher than or equal to 62 mg/kg body weight. This loss of early spontaneous proliferation coincided with the disappearance of medium-EPM thymocytes. By contrast, proliferative response of thymocytes to mitogens was enhanced by OHC pretreatment of donors and roughly paralleled the enrichment in high-EPM cells. However, administration of the largest amounts of OHC diminished this mitogen responsiveness, although PHA reactivity was less affected than Con A reactivity.

Surface modification and electrophoresis of normal and transformed BHK21 cells.

ABSTRACT The mean electrophoretic mobilities at pH 7·5 of virus-transformed (Py6) and normal BHK21 cells were very similar, whether they were harvested mechanically or by the use of trypsin. After formaldehyde treatment, there was significantly increased mobility in both cell types; the transformed cells showed significantly the greater change. After neuraminidase treatment, the mean electrophoretic mobility was decreased to the same extent in both types of cell. Treatment with neuraminidase and formaldehyde had no effect on the mean electrophoretic mobility of the normal cells but slowed the transformed variety. The mobility in histone solution had no relationship to histone concentration but was statistically correlated with the amount of histone per cell, calculated from total histone present divided by the total number of cells; a linear relationship being obtained with the normal cells but an initial plateau was demonstrated with the transformed cells. The normal cell line showed a similar plateau after neuraminidase treatment. The possible significance of these results is discussed.

Destruction and regeneration of lymphocyte populations in the mouse spleen after cyclophosphamide treatment.

Three parameters allowing to distinguish B and T lymphocyte populations, i.e., the presence of surface-associated immunoglobulins (slg), the electrophoretic mobility (EPM) and the responsiveness <i>in vitro </i>to bacterial lipopolysaccharide (LPS) and to concanavalin A (Con A), were studied on mouse spleen cells at various time intervals following a single injection of cyclophosphamide (300mg/kg body weight). 1 day after the treatment, B cells (sIg-bearing, slow EPM) were almost totally destroyed and likewise, the response to LPS was abolished. Regeneration of the B cell compartment did not begin before day 16 and was complete only by day 30. Reconstitution of the LPS responsiveness was found to parallel the reappearance of B cells. T cells were also affected by the treatment, although to a much lesser extent than B cells. A first T cell subpopulation, with a mean EMP = 1.10 <i>μ</i>msec<sup>––</sup><sup>1</sup> V<sup>––1</sup> cm, was destroyed by cyclophosphamide, while a large part of the second T cell subpopulation, with a mean EPM = 1.25 <i>μ</i>m sec<sup>––1</sup> V<sup>––1</sup> cm remained in the spleen. This strongly suggests that the former subpopulation probably corresponds to short-lived cells and the latter to long-lived ones. Con A responsiveness was diminished but never suppressed and recovered relatively early. Starting by day 8, complete regeneration of the T cell compartment was achieved by day 30.

Electrophoresis of Blood Platelets and Erythrocytes: A Study in Healthy Persons and Patients with Haematological Diseases, Prosthetic Heart Valves, Virus Infections and Hyperlipaemia

The mean electrophoretic mobility of normal human blood platelets in 301 samples from 260 healthy individuals was 1.08 μm/s/V/cm in citrated plasma and 0.84 and 1.05 μm/s/V/cm in 0.145 M saline (pH 7.3) and in sucrose/phosphate/saline buffer (pH 6.4), respectively. Normal red cell mobility in citrated plasma in 229 samples from 195 persons was 1.41 μm/s/V/cm, in 0.145 M saline and 0.0667 M phosphate buffer (pH 7.3) 1.09 and 1.28 μm/s/V/cm, respectively.The results of blood platelet and red cell electrophoresis in 90 patients with various diseases are reported. Severe abnormalities in platelet mobility were not found in any of the patients. In patients with von Willebrand's disease a slight increase in platelet mobility was observed. In 4 patients with haemolytic‐uraemic syndrome and 2 patients with microangiopathic haemolytic anaemia a reduced red cell mobility in citrated plasma and buffers was found.In a case of red cell polyagglutinability, two populations of erythrocytes were observed electrophoretically. About half of the red cells had a mobility of about 43 % of normal in citrated plasma.

Electrophoresis of lymphoid cells. Organ specific distribution pattern of small lymphoid cells in the mouse.

The electrophoretic mobility of small lymphoid cells in the thymus, lymph nodes, and spleen was measured. The distribution patterns were organ specific. The majority of the thymic cells were slow with a small ‘tail’ of faster cells. Both lymph node and spleen cells showed a fast and a slow cell population with a difference in mean electrophoretic mobility of about 40%. The percentage of cells to the fast and slow groups was similar to the percentage of T‐ and B‐cells in these organs It is therefore suggested that T‐and B‐cells may be characterized by their different electrophoretic mobilities and also that there may be a change in the net surface charge during maturation from thymocyte to T‐lymphocyte.

Detection of differences in surface-charge-associated properties of cells by partition in two-polymer aqueous phase systems.

WHEN aqueous solutions of two polymers are mixed in certain proportions they may form two-phase systems1,2 which can be buffered and used to partition and separate cells, particles and macromolecules by countercurrent distribution (CCD). Partition generally depends on polymer composition and concentration, the ionic composition and the charge sign of the material being partitioned. Such systems have been used to separate erythrocytes from white cells and erythrocytes on the basis of age. Changes in the surface properties of cells resulting from enzyme treatment or storage have also been demonstrated by this means3. Higher cell partition often accompanies increasing electrophoretic mobility which suggests that surface charge may be an important factor in partitioning4–6. An apparent exception to this is the increased partition of stored human erythrocytes as compared with fresh7, as opposed to the mean electrophoretic mobility of both cell populations which remain identical8.

PHYSICAL HETEROGENEITY OF BOVINE GAMMA GLOBULINS: GAMMA-1M GLOBULIN ELECTROPHORETIC HETEROGENEITY.

SummaryIn the bovine species, gamma-1M globulin occurs as an electrophoretic continuum with most molecules of near mean mobility. No electrophoretic sub-groups of particularly high concentration, as described for 7S gamma globulins, were noted. Complement-fixing antibody activity was associated with bovine gamma-1M globulin molecules whose mean electrophoretic mobility was slower than the mean mobility of the protein as a whole. The marginal extent of this manifestation argues against anticipation of as heterogeneous a distribution of particular biological activities within the gamma-1M globulin spectrum as has been shown to be the case with 7S gamma globulins.