Inhibition Of Cell Aggregation Research Articles

The role of membrane lectins in Dictyostelium discoideum aggregation as ascertained by specific univalent antibodies against discoidin I.

Antibodies against pure discoidin I have been used as a tool to ascertain the role of this lectin in aggregation of Dictyostelium discoideum. Discoidin I is widely expressed over the cell surface of aggregation-competent AX-2 cells, as ascertained by indirect immunofluorescence with specific (antidiscoidin I) antibodies. Univalent antidiscoidin I antibodies (Fab fragments) inhibit the aggregation-specific intercellular adhesion of D discoideum AX-2 cells in an in vitro assay. This inhibition depends on antibody concentration and cell density; a 50% inhibition of cell aggregation was obtained at antidiscoidin I Fab concentration of 4.5 mg/ml and 1 X 10(6) cells/ml. Aggregation and morphogenesis on solid support is also effectively inhibited when AX-2 cells are starved in the presence of antidiscoidin I Fab fragments. The inhibition of morphogenesis is also dose dependent and more effective than in the in vitro assay. No inhibition of aggregation either in the in vitro assay or on morphogenesis on solid support was observed with preimmune Fab fragments at any of the concentrations tested (up to 9.6 mg/ml).

Flocculation in yeast, an assay on the inhibition of cell aggregation

Flocculation in yeast depends on the presence of bivalent ions such as Ca++ causing cell aggregation. By binding the Ca++ with EDTA this aggregation may be inhibited. The amount of EDTA to prevent aggregation is strain-specific. It is called the flocculation value and expressed in micromoles EDTA. The flocculation value may be used to characterize both sedimenting and floating yeasts.

Inhibition of cell aggregation by vanadate

Inhibition of cell aggregation by vanadate

Aggregation patterns of fetal rat brain cells following exposure to X-irradiation.

In our search for a simplified in vitro test system to assess the teratogenic effects of physical factors, we studied the effects of total maternal body X-irradiation on aggregation patterns of enzymatically isolated fetal rat brain cells and on ultrastructural aggregate changes. The fetal brain cells were derived from day 14 gestation fetuses of pregnant Sprague-Dawley (CD strain) rats exposed to X-irradiation (25 - 200 R) one hour prior to sacrifice. Notable changes in the cell aggregates following X-irradiation included a reduction in cell aggregate size and an increase in number. The frequency of cell aggregates was higher in the treated than in the control group, and the mean diameter of cell aggregates was inversely related to increasing X-irradiation doses. Transmission electron microscopy revealed in isolated cells features of degenerative process which were similar to those found in intact fetal brain lesions caused by maternal X-irradiation. Furthermore, scanning electron microscopy revealed that inhibition of cell aggregation following X-irradiation could probably be attributed to inhibition of membrane filopodia development and a consequent failure fo cell aggregates to fuse into a greater cell aggregate mass. These results suggest that the membrane factors which influence cell aggregation may be a useful parameter to assess early effects of X-irradiation-induced brain deformity. Presently, the cell aggregation culture system is being further evaluated as a short term test system for environmental teratogens.

Chymostatin inhibits cellular aggregation of activated human peripheral blood lymphocytes

Activation of lymphocytes with phytohaemagglutinin (PHA) induces cell aggregation (CA). Although the events leading to CA are not clear, a general change in protein turnover has been observed. Therefore, we have examined the effect of some well characterized protease inhibitors on CA. Chymostatin (20–150 μM) produced a dose dependent inhibition of CA. Protease inhibitors other than chymostatin had no significant effect on CA. Based on the action of these protease inhibitors on CA and their known inhibitory effects on different proteases, it is suggested that chymotrypsin or a chymotrypsin-like activity may be involved in PHA-stimulated aggregation of human peripheral blood lymphocytes.

Inhibition of cell aggregation by specific carbohydrates

One approach to investigating the potential role of surface carbohydrates in mediating intercellular adhesion is to study cell reaggregation in the presence of defined concentrations of specific saccharides. Fifteen different exogenously added saccharides were tested for their effect on the reaggregation of 24 h sea urchin embryo cells ( Strongylocentrotus purpuratus) dissociated by removal of divalent cations. Aliquots (0.2 ml) of cell suspension were rotated at 68 rpm, 17 °C, pH 8.0, with varying concentrations (0.5 × 1 −1−0.5 × 10 −5 M) of the sugars. Relative percents of cell aggregation were determined using an electronic particle counter assay. In all experiments cell viability using trypan blue was over 95.8%. Among the sugars tested, in 15 separate experiments, d-galactose and N-acetyl- d-galactosamine consistently inhibited aggregation to the greatest extent at early time points. d-Galactose, at all concentrations tested, at 10, 20, 30, 40, and 60 min rotation, showed mean decreases of aggregation over control values in the absence of sugar of 59.3, 53.6, 43.2, 35.0 and 36.4%, respectively. N-Acetyl- d-galactosamine also caused mean decreases in aggregation of 73.5, 54.5, 40.8, 42.2 and 45.6%, respectively. Each difference over the control is significant to the p value of less than 0.01. In three experiments, β-galactosidase substantially inhibited reaggregation of these cells. These results suggest that galactopyranosyl-like groups may be implicated in mediating adhesion of 24 h sea urchin embryo cells to each other.

Concanavalin a and wheat germ agglutinin receptors on dictyostelium: Their visualization by scanning electron microscopy with microspheres

The cellular slime mold, Dictyostelium discoideum, is a convenient model for studying cellular interactions during development. Evidence that specific cell surface components are involved in cellular interactions during its development has been obtained by Gerisch and co-workers (1, 2) using immunological techniques. Smart and Hynes (3) have shown that a cell surface protein can be iodinated on cells in aggregation phase, but not in vegetative phase, by the lactoperoxidase procedure. Recently, McMahon et al. (4), and Hoffman and McMahon have demonstrated, by SDS gel electrophoresis, considerable differences in cell surface proteins and glycoproteins of plasma membranes isolated from cells at different stages of development. Plant lectins have also been used to monitor changes in cell surface properties of D. discoideum cells during development. Weeks and co-workers (5, 6) have detected differences in the binding and agglutination of cells by concanavalin A (Con A). Gillette and Filosa (7) have shown that Con A inhibits cell aggregation and prematurely induces cyclic AMP phosphodiesterase. Capping of Con A receptors has also been reported (8). Reitherman et al. (9) have recently reported that agglutination of cells by several plant lectins and the slime mold agglutination, discoidin, changes during development. Such studies indicate that differences in surface properties exist for cells at various stages of development. However, owing to the uncertainties in the factors which contribute to lectin-induced cell agglutination (10), the molecular basis for these observations remain to be determined. In this study, we have used microspheres (11-14) coupled to either Con A or wheat germ agglutinin (WGA) as visual markers to study by scanning electron microscopy the topographical distribution of lectin receptors on D. discoideum cells fixed at different stages of development. We also describe the effect of labeling on the distribution of lectin receptors and on the morphology of the cell surface.

Bhk21 fibroblast aggregation inhibited by glycopeptides from the cell surface

Glycopeptides were removed by trypsinization from the surface of baby hamster kidney cells (line BHK21-C13), digested by pronase and separated into 2 fractions by exclusion chromatography. The addition of small amounts of either glycopeptide fraction to shaken suspensions of lightly trypsinzied cells inhibited their rapid aggregation, but one fraction was more active than the other and in higher concentrations it was able to inhibit aggregation completely. After this fraction was purified by high-voltage electrophoresis one subfraction also inhibited aggregation. The effect of the glycopeptides increased following their pretreatment with neuraminidase, but preincubation with periodiate or galactose oxidase destroyed all activity. Galactose oxidase also inhibited cell aggregation directly. Similar glycopeptides from virus-transformed BHK21 cells, oligosaccharides and intact and desialysed human urinary glycoproteins had comparatively little or no effect on BHK21 cell aggregation. The results suggest terminal beta-galactosides and possible alpha-galactosides, and to some extent a particular substructure of cell surface heteroglycans are necessary for their inhibitory activity. The parent, plasma membrane of glycoproteins might serve as adhesive binding sites in cell cohesion, but some evidence indicates cell surface sialyl- and galactosyltransferases may not ordinarily act as their complementary binding receptors.

Cell aggregation: properties of cell surface factors from five species of sponge.

AbstractAggregation factors isolated from the cell surface of five species of sponge are shown to be specific in their ability to enhance cell aggregation. Factors from Haliclona viridis, Haliclona variabilis, Tedania ignis, Homaxinella rudis, and Dysidea crawshayi each exhibit the species specific enhancing effect and have no such effect in heterospecific cell‐factor combinations. Radioactively labeled cell free supernatant materials are shown to bind selectively to homospecific cells suggesting that cells contain specific receptors for attachment of the aggregation mediating materials. Finally, in several heterospecific combinations there was an inhibition of cell aggregation. The inhibitory agent was separated from the enhancing factor chromatographically. After removal of the inhibitory substance, the enhancing factor was still active in promoting cell aggregation of the homospecific cells only.

Evidence for the importance of puromycyl peptides in the inhibition by puromycin of cell aggregation in vitro.

ABSTRACT Trypsin-dissociated cells from the muscle tissue of 9-day-old chick embryos were employed to investigate the effects of cycloheximide and a puromycin-cycloheximide mixture on cell aggregation, protein synthesis and respiratory metabolism. Cycloheximide when introduced at a concentration of 10 μg/ml into a suspension of cells in Eagle’s MEM inhibited aggregation by 25 % at 24 h. At this time an inhibition of 40 % was apparent in the presence of a mixture of cycloheximide and puromycin both at a concentration of 10 μg/ml. Both cycloheximide and the cycloheximide-puromycin mixture arrested protein synthesis of rotated cells by 90 % within 15 min of introducing the antibiotics into cell suspensions. The antibiotics retained their inhibitory effects on protein synthesis for the 24-h period of rotation. Cycloheximide inhibited cellular oxygen uptake and carbon dioxide evolution of rotated cells by 25 % at the end of the 24-h experimental period. At this time an inhibition of 30 % was observed in the presence of the cycloheximide-puromycin mixture. The release of radioactive carbon dioxide by cycloheximide-treated cells was inhibited by 46 % at 24 h. In the presence of the antibiotic mixture, 14CO2 release was inhibited by 30 % at 4 h, but after 8 h very little further 14CO2 was evolved. As a control, puromycin (10μg/ml) inhibited cell aggregation and respiration to an extent similar to that previously reported. The results are discussed in terms of puromycyl peptides producing a metabolic effect on cell aggregation. It is considered that this is additional to the effect of puromycin inhibiting aggregation through the arrest of protein synthesis.

A biochemical investigation into the inhibitory effects of glucosamine and N-acetylclucosamine on the aggregation in vitro of embryonic chick muscle cells.

ABSTRACT The effect of glucosamine and N-acetylglucosamine on aggregation and energy metabolism was investigated over an 8-h period in cells dissociated by 0·25 % (w/v) trypsin from the skeletal muscle of 9-day-old chick embryos. At 8 h, 0·023 M glucosamine and N-acetylglucosamine inhibited the aggregation of cells suspended in Eagle’s minimal essential medium by 18·9 % and 16·4% respectively, as judged on a basis of aggregate size. Glucosamine and N-acetylglucosamine reduced the cellular ATP level by a mean of 36 % and 27 % respectively; values reflected in the 32% and 19% loss of total adenine nucleotides caused by these sugars. The adenine nucleotide balance was also changed from a mean control ATP/AMP ratio of I3·7 to 8·75 by N-acetylglucosamine and to 8·0 by glucosamine. Intracellular lactate/pyruvate ratios were similarly disrupted in cells incubated with 0·023 M hexosamine. Although the hourly values fluctuated, it was seen that the amount of lactic acid relative to pyruvic acid, considered as an average for the 8-h period, was raised from 6:1 in controls to 8:1 in N-acetylglucosamine-treated and to 10:1 in glucosamine-treated cell preparations. Compared to controls at 8 h, glucosamine enhanced the production of lactate into the suspension medium by 99%. The N-acetyl analogue caused cells to produce more lactic acid than did controls for 4–5 h only, for by 8 h 25 % less of this metabolite was assayed in the culture medium. The incorporation of D-[U-14C]glucose into glycogen paralleled the results of extracellular lactic acid assays. N-acetylglucosamine inhibited the incorporation by 30 % at 4 h, although by 6 h, and for the remainder of the experimental period, there was more 14C-labelled glycogen in these cells than in controls. By contrast, glucosamine inhibited the incorporation of radioactive glucose into glycogen by 42% at 4 h and, unlike N-acetylglucosamine, consistently thereafter. Glucosamine also enhanced cellular oxygen uptake throughout the experimental period, to the extent of 59 % at 8 h. The oxygen uptake of N-acetylglucosamine-treated cells was similar to controls until about the 5th hour, when there was a subsequent inhibition which had accumulated to 13 % by the end of the experiment. The release of 14COs by cells was inhibited by glucosamine. This hexosamine depressed production by 19% at 12 h whereas N-acetylglucosamine inhibited this evolution by 9 % at this time. The metabolic effects of these hexosamines on chick muscle cells in vitro are mainly attributed to a central alteration of the adenine nucleotide balance although certain other documented effects of glucosamine are considered to be involved. An inhibition of cell aggregation by glucosamine and N-acetylglucosamine is discussed in terms of a depressed cellular metabolic economy.

Studies on the mechanism underlying the inhibition by puromycin of cell aggregation in vitro.

ABSTRACT Cells dissociated with 0·25 % crude trypsin from the muscle tissue of 9-day-old chick embryos were employed to investigate the effect of puromycin on cellular metabolism. Parallel studies were also made, using the gyratory shaker, to confirm the effectiveness of puromycin in inhibiting cell aggregation and protein synthesis. Puromycin when introduced at a concentration of 10μg/ml into a suspension of cells in Eagle’s MEM did not completely inhibit cell aggregation. Small aggregates were formed in the first 4 h of the experiment. Protein synthesis of the rotated cells, as measured by the incorporation of L-[α -14C]leucine into proteins, was arrested by 91 · 7% within 15 min of introducing puromycin into a cell suspension. The antibiotic retained its inhibitory effect on protein synthesis for the 24-h period of rotation. Puromycin inhibited the cellular oxygen uptake and carbon dioxide evolution of the rotated cells by 40 % within 4 h of its introduction. However, treated cells were still respiring, though at a much reduced rate, at the end of the 24-h experimental period. The release of radioactive carbon dioxide by puromycin-treated cells was also inhibited by 40% at the 4-h stage but after 8 h no further14CO2 was evolved. The presence of the antibiotic markedly inhibited the uptake of glucose by trypsin-dissociated cells. The level of glycogen and lactate in cells suspended in Eagle’s MEM was reduced very considerably over a 24-h period. The presence of puromycin accelerated glycogen utilization over the first 6 h of rotation but at 24 h there was a difference of only 0 · 6 % between the glycogen content of treated cells and controls. At 24 h 11·3 % less lactate remained in the puromycin-treated cells than in the controls. The ATP/ADP ratio of trypsin-dissociated cells decreased from an initial value of 2 · 59 to 1 · 45 after rotation for 24 h. In the presence of puromycin the ATP/ADP ratio was 0 · 62 at 4 h and had further declined to 0 · 48 by 24 h. The effects of puromycin on the aggregation, protein synthesis and cellular metabolism of trypsin-dissociated cells are discussed in relation to cellular adhesive mechanisms.

Inhibition of cell aggregation by antibodies directed against actomyosin.

IT has been proposed that cell adhesion involves the participation of actomyosin-like protein located in the surface1. The theory was based mainly on the similarity between actomyosin proteins in muscle2–5 and non-muscle cells6–8, a comparison of the properties of actomyosin protein with the effects of calcium and of nucleoside phosphates9–11 on cell aggregation, and evidence indicating the presence of actomyosin-like protein at the surface of liver cells12–15. Hypothetical models have been suggested for the involvement of contractile actomyosin in inter-molecular bonding between adjacent cells16–18.

Aggregation of dissociated embryonic chick cells at 3 degrees.

WHILE investigations of the effects of environmental factors on aggregation in vitro of dissociated embryonic cells were under way in this laboratory1, Curtis reported2 that “extensive visible coherent re-aggregation” occurred at 3° C in suspensions of embryonic chick limb bud cells in a medium without serum. He concluded that the cells were able to aggregate normally at low temperatures, and that horse serum inhibited cell aggregation at sub-optimal temperatures. These conclusions differed from the previously demonstrated3,4 and corroborated5 temperature-dependence of aggregation of trypsin-dissociated embryonic cells. Because versene (EDTA) was the dissociating agent used in Curtis's experiments, the possibility arose that, in contrast to trypsin-dissociated cells3–5, cells dissociated with versene might be able to aggregate normally at low temperatures; since such differences in aggregation behaviour between differently dissociated cells would be of obvious interest and usefulness in studies on cell adhesiveness, attempts were made to repeat and extend Curtis's results, using his experimental procedures.

Inhibition of cell aggregation in vitro by puromycin

Inhibition of cell aggregation in vitro by puromycin

The inhibition of cell aggregation by a pure serum protein

ABSTRACT The aggregation of isolated cells into coherent multicellular bodies is widely thought to be due mainly if not entirely to the adhesiveness of the cells for one another, according to Moscona (1961a, b), Curtis (1962) and Steinberg (1962a) amongst others. In consequence the aggregation of cells from dispersed (disaggregated) tissues has been widely used as a test for the degree of adhesiveness shown by the cells, and conditions affecting aggregation have been interpreted as affecting cell adhesion. Using this type of test Moscona (1961a, b) found that embryonic chick cells would not aggregate at temperatures below 14° C. It was also discovered that aggregation was inhibited at 37° C. by puromycin and actinomycin D (Moscona & Moscona, 1963), by glucosamine-HCl (Garber, 1963), and by chloramphenicol (Nakanishi et al., 1963). Moscona concluded from the failure of aggregation at low temperatures that the metabolic synthesis of an adhesive substance was being prevented under such conditions and this interpretation was reinforced by the evidence of chemical inhibition of aggregation. Moscona (1962) suggested that the adhesive substance was responsible for producing cell to cell adhesion. In all these experiments cells were aggregated in media containing serum, usually horse serum.

Zellfunktionen und zellfunktionswechsel in der entwicklung von Dictyostelium discoideum: VI. Inhibitoren der aggregation, ihr einfluss auf zellkontaktbildung und morphogenetische bewegung

Inhibition of cell aggregation by 2,4-dinitrophenol (DNP), 2-mercaptoethanol (MCAe) and urethan was tested with amoeba cells of Dictyostelium discoideum. The following inhibitory effects on aggregative cell movements and intercellular contacts have been observed. 1. 1. All substances tested inhibit aggregation by inhibition of cell movement. 2. 2. Moreover, DNP reversibly interferes with the activity of contractile vacuoles and with protoplasmic streaming; cells in any developmental stages are sensitive. Since similar effects can be produced by anaerobic conditions, it is concluded that in the material used in these experiments, oxidative phosphorylations are indispensable for fundamental cellular functions. Thus, DNP has not a specific action as an inhibitor of aggregation. 3. 3. MCAe action also yields the cells spherically, but the other characteristic effects of DNP are not observed. It is assumed that MCAe among others acts on the cell membrane directly. 4. 4. Formation of intercellular contacts is not reduced drastically by DNP; cell substrate contacts, on the contrary, are inhibited (nearly) completely. 5. 5. MCAe considerably weakens the non phase-specific intercellular contacts which are characterized by high EDTA-sensitivity. The cell binding specific for cells in the aggregation stage is reduced but little by MCAe. It is noticed that the cellular basis of Dictyostelium development in some respects is quite similar to that of more complicated developmental systems in animals.