Quantitative estimation of cell aggregation in short time culture.
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Animals↗
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Results for “Cell Aggregation”
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Immunohistochemical localization of alpha-keratin and fibronectin in skin cell aggregates.
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Animals↗
Stimulation of prolactin release by angiotensin II in superfused rat anterior pituitary cell aggregates.
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Angiotensin II↗
Electron microscopy of a nevus cell aggregate associated with an axillary lymph node.
The cellular composition of the so-called nevus cell aggregate (NCA) or inclusion has been the subject of controversy. Although the predominant opinion is that these cells are neval, some investigators suggest that they are related to glomus cells. In order to investigate this further, an NCA in the capsule of a nontumorous axillary lymph node obtained from a patient with breast cancer was removed from a paraffin block and processed for electron microscopic study. Ultrastructurally, uniform, round nevus cells with indented nuclei, dispersed chromatin, randomly oriented cytoplasmic fibrils, and a few mature melanosomes were identified. These cells closely resembled cutaneous intradermal neval cells and appeared unrelated to glomus cells. It is postulated that these NCA could be a noncutaneous anatomic source of some malignant melanomas.
Aged↗
A novel Dictyostelium discoideum gene required for cAMP-dependent cell aggregation.
Using a method of random insertional mutagenesis called REMI (restriction enzyme-mediated integration), we isolated two mutant strains of Dictyostelium discoideum with a defect in cAMP-dependent cell aggregation. On bacterial lawns, both of the cells formed large and smooth plaques. When starved in a non-nutrient medium, they became elongated and extended pseudopods very frequently like starved wild type cells. However, they never formed streams toward an aggregation center. Genomic DNA fragments flanking the sites of insertion of the REMI tag were rescued from the mutant cells. The fragments contained one common open reading frame encoding a protein of 1148 amino acid residues. The protein's sequence is homologous to those of two hypothetical proteins of S. cerevisiae and S. pombe.
Amino Acid Sequence↗
[Mode of action of a diffusable factor facilitating cancer cell aggregation in vitro: effect at +6 degrees C. on mechanically dissociated cells].
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Animals↗
Evidence that folliculo-stellate cells do not impede the permeability of intercellular spaces to molecular diffusion in three-dimensional aggregate cell cultures of rat anterior pituitary.
The permeability of intercellular spaces within the anterior pituitary (AP) and the influence of folliculo-stellate (FS) cells on compartmentalization within this tissue, has become a matter of debate. In reaggregated pituitary cell cultures as well as in the AP in situ the intercellular gaps and follicle-like structures remain accessible to molecular diffusion, whereas in some studies FS cells were reported to form tight epithelia that impede macromolecular transport through the spaces between the epithelial cells. In the present study the permeability of AP cell reaggregates was examined using fluorescent BSA as a tracer. Using confocal scanning laser microscopy a direct visualization of the permeation process was achieved. Quantitative estimation of the effective diffusion coefficient (Deff) for fluorescein-BSA within the aggregates was obtained using the fluorescence photobleaching recovery technique. Deff was 1.33 +/- 0.31 x 10(-7) cm2/sec (mean +/- SD) in aggregates from 14-day-old female rats and 2.45 +/- 0.55 x 10(-7) cm2/sec in aggregates from adult female rats. These values are about three times lower than in free solution. Calculation of the time-dependent concentration distribution inside the aggregate for a Deff = 2 x 10(-7) cm2/sec revealed that the concentration of the fluorescent tracer in the center of the aggregate reaches 90% of the concentration outside the aggregate after 0.5 min for aggregates with a radius of 50 microns and 6 min for aggregates with a radius of 150 microns. Aggregates enriched in FS cells, in which we previously showed a sustained inhibition of secretory responses to stimulatory and inhibitory agents as compared to total population aggregates, showed a diffusion coefficient (Deff = 1.85 +/- 0.77 x 10(-7) cm2/sec) which was not significantly different from that in the total population aggregates. The present study shows that AP cell aggregates are fully permeable to diffusing molecules within minutes and that in a three-dimensional tissue configuration FS cells, which were reported to tonically inhibit AP hormone release in response to various secretagogues, do not impede molecular diffusion to an extent which would account for sustained inhibition of hormone release.
Animals↗
Models of monocytic invasion into glioma cell aggregates.
BACKGROUND: In order to investigate why human gliomas are abundantly infiltrated by monocytic cells without signs of antitumor activity, experimental models were established in vitro and in vivo. MATERIALS AND METHODS: Peripheral human blood monocytes were added to A172 or U118 glioma cell spheroids and probes analyzed after 72 h by immunohistochemistry. Fluorescence-labelled peritoneal macrophages were administered to syngeneic RG2-glioma-bearing Fischer rats by intravenous or intracarotid injection. RESULTS: Spheroids of both cell lines were infiltrated by monocytes, which took on a chronic inflammatory phenotype with co-expression of MRP8 and MAC 387/MRP14 and positivity to 25F9, but not to 27E10. After both intra-arterial and intravenous injection, labelled monocytes accumulated within the tumor parenchyma of the rat gliomas, while the surrounding brain was only sparsely infiltrated. CONCLUSION: The experimental models described here allow for further investigation of the interactions between monocytes and glioma cells, both in vitro and in vivo. Moreover, monocytes that infiltrate from the peripheral blood into brain tumors may serve as carriers for targeted therapies.
Animals↗
A role for sulfated polysaccharide recognition in sponge cell aggregation.
Molecules binding sulfated polysaccharides were detected as lectins in cholate lysates of cells from twelve sponge species. Each species exhibited a unique binding profile. The pattern of binding indicated that the specificity was most probably determined by the orientation of the sulfate groups on the polysaccharide chains. Cells from each of the three species examined in more detail were found to express sulfated polysaccharide-binding molecules at their surface and at least one of the polysaccharides recognized was found to inhibit the reaggregation of cells from each species. Moreover, in all but one instance, lectins for the inhibitory polysaccharide were both detected in cell lysates and shown to be expressed at the cell surface. Sulfated polysaccharides, therefore, appeared to be involved in cell interaction events in the Porifera. This conclusion was confirmed by the isolation via ion exchange chromatography of an endogenous polysaccharide from an O. tenuis cell extract. This molecule contained uronic acid and hexose units in a ratio of 2:1, 11.9% sulfur and less than 0.5% protein. It inhibited the aggregation of O. tenuis cells and the agglutination of dextran-sulfate- and polyvinyl-sulfate-coupled erythrocytes by O. tenuis cell lysates. O. tenuis cell aggregation was also inhibited by polyvinyl sulfate and dextran sulfate and molecules binding these compounds were expressed on the surface of O. tenuis cells. Thus, is was probable that the cell surface receptor for polyvinyl sulfate and dextran sulfate and isolated sponge sulfated polysaccharide are one and the same. Finally, using a dextran sulfate affinity procedure, a 35 kD dextran-sulfate-binding protein was isolated from the surface of O. tenuis cells. The possibility that the polysaccharide isolated from O. tenuis cell extracts in the absence of calcium is the monomeric form of a cell aggregation-enhancing factor is discussed.
Animals↗
Effects of the ethyl acetate fraction of Spatholobi caulis on tumour cell aggregation and migration.
The ethyl acetate (EA) fraction obtained from a methanol extract of Spatholobi caulis (Leguminosae) has been investigated for anti-metastatic activities in vitro. The EA fraction of Spatholobi caulis inhibited platelet aggregation induced by B16BL6 melanoma cells with an IC(50) of 50 microgram/mL. The EA fraction significantly inhibited HT1080 cancer cell invasion through a matrigel-coated filter with an IC(50) of 25 microgram/mL. Messenger RNA expression of uPA was effectively decreased in HT1080 cells by the EA fraction of Spatholobi caulis with an IC(50) of 30 microgram/mL, but the expressions of MMP-2 (matrix metalloproteinase) and TIMPs (tissue inhibitors of metalloproteinases) were not changed. These findings indicated that the EA fraction suppressed tumour cell invasion by downregulation of uPA (urokinase-type plasminogen activator). Taken together, these results suggest that the EA fraction of Spatholobi caulis may have anti-metastatic activities by blocking tumour cell-induced platelet aggregation (TCIPA) and tumour cell invasion.
Animals↗
Whole blood white cell aggregation: a novel technique.
Epidemiological studies have suggested that subjects who develop ischaemic events have an elevation in their numbers of white blood cells. White cells are similar to platelets in that they can adhere, aggregate and undergo a release reaction. We wished to investigate white cell aggregation in vascular diseases, and have developed a new technique to measure whole blood white cell aggregation. This paper describes the method with special reference to the aggregating agents used. The effect of Iloprost, a synthetic prostacyclin analogue, on white cell aggregation has also been investigated.
Adult↗
A model for motion and sedimentation of cylindrical red-cell aggregates during slow blood flow in narrow horizontal tubes.
When blood flows slowly in a narrow tube, red-cell aggregation results in formation of an approximately cylindrical "core" of red cells, which moves as a rigid body. The core is denser than the surrounding fluid, and sedimentation is observed in horizontal tubes. To model this, the Stokes flow of a fluid surrounding a long solid cylinder (the core) contained in a long hollow cylinder (the tube) is considered. The cylinder axes are parallel but not coincident. An exact analytic expression for the resistance coefficient for motion perpendicular to the axes is given. This coefficient increases rapidly with the ratio of core radius to tube radius, and core eccentricity. The predicted rate of sedimentation is comparable to that observed experimentally. The apparent viscosity of a two-phase medium consisting of a core of aggregated particles and surrounding pure fluid is calculated. For a core radius corresponding to experimental conditions, the apparent viscosity increases rapidly with increasing eccentricity of the core.
Blood Flow Velocity↗
Voltage clamp analysis of embryonic heart cell aggregates.
The double-microelectrode voltage clamp technique was applied to small spheroidal aggregates of heart cells from 7-d chick embryos. A third intracellular electrode was sometimes used to monitor spatial homogeneity. On average, aggregates were found to deviate from isopotentiality by 12% during the first 3--5 ms of large depolarizing voltage steps, when inward current was maximal, and by less than 3% thereafter. Two components of inward current were recorded: (a) a fast, transient current associated with the rapid upstroke of the action potential, which was abolished by tetrodotoxin (TTX); and (b) a slower inward current related to the plateau, which was not affected by TTX but was blocked by D600. The magnitudes, kinetics, and voltage dependence of these two inward currents and a delayed outward current were similar to those reported for adult cardiac preparations. From a holding potential of -60 mV, the peak fast component at the point of maximal activation (-20 mV) was -185 microA/cm2. This value was about seven times greater than the maximal slow component which peaked at 0 mV. The ratio of rate constants for the decay of the two currents was between 10:1 and 30:1.
Action Potentials↗
Myosin heavy chain kinase inactivated by Ca2+/calmodulin from aggregating cells of Dictyostelium discoideum.
Soluble myosin heavy chain kinases (MHC kinases) were partially purified from growth phase and aggregation-competent cells of Dictyostelium discoideum. In the aggregation-competent cells, two MHC kinases were distinguishable. One of these enzymes, called MHC kinase II, was inactivated by Ca2+ and calmodulin in a highly temperature-dependent reaction. A MHC kinase found in growth phase cells did not have these regulatory properties. Substrate specificities were analysed for MHC kinase II and for the MHC kinase from growth phase cells. Both enzymes phosphorylated threonine residues of the myosin heavy chains of D. discoideum and Physarum polycephalum. Phosphopeptide mapping of D. discoideum myosin and determination of the stoichiometry of its phosphorylation suggested the presence of two phosphorylation sites per heavy chain. Both sites were contained within a 38-kd chymotryptic fragment. The inactivation of MHC kinase II by Ca2+ plus calmodulin suggests this enzyme has a role in the regulation of myosin functions during the chemotactic response of a cell. The phosphorylated myosin had about one third the actin-activated Mg2+-ATPase activity of the non-phosphorylated myosin. Previous findings indicated that stimulation of D. discoideum cells with the chemo-attractant cAMP increases the cytoplasmic Ca2+ concentration. Under these conditions MHC kinase II might be inhibited and the dephosphorylated, more active form of myosin would accumulate.
Calcium↗
Mutants of Polysphondylium pallidum altered in cell aggregation and in the expression of a carbohydrate epitope on cell surface glycoproteins.
Mutants of the cellular slime mold Polysphondylium pallidum have been selected using a cell sorter and a fluorescentlabeled monoclonal antibody, mAb 293. This antibody blocks cell adhesion when applied as Fab, and recognizes a carbohydrate epitope containing L-fucose. This epitope is expressed on the cell surface and is present on >10 membrane glycoproteins of different apparent mol. wts. Twenty mutants were obtained which did not bind mAb 293 when tested at 2 h of starvation. After longer periods of starvation the epitope became detectable in the mutants. In all these mutants aggregation patterns were atypical. Generally streams of cells that were radially orientated around aggregation centers were missing or were much shorter than in wild-type. Genetic analysis demonstrated that aberrant aggregation was linked to the alteration in carbohydrate epitope expression. One mutant was unstable and gave rise to subclones in which almost no antibody binding was observed, even after 24 h of starvation, and only few aggregation centers with no streams or very short ones were formed. These results indicate that the capability of the cells to aggregate is correlated with the exposure on their surfaces of the carbohydrate epitope recognized by mAb 293, whose function in development remains to be established.
Journal Article↗
Selective neurodegeneration induced in rotation-mediated aggregate cell cultures by a transient switch to stationary culture conditions: a potential model to study ischemia-related pathogenic mechanisms.
Aggregating brain cell cultures at an advanced maturational stage (20-21 days in vitro) were subjected for 1-3 h to anaerobic (hypoxic) and/or stationary (ischemic) conditions. After restoration of the normal culture conditions, cell loss was estimated by measuring the release of lactate dehydrogenase as well as the irreversible decrease of cell type-specific enzyme activities, total protein and DNA content. Ischemia for 2 h induced significant neuronal cell death. Hypoxia combined with ischemia affected both neuronal and glial cells to different degrees (GABAergic neurons>cholinergic neurons>astrocytes). Hypoxic and ischemic conditions greatly stimulated the uptake of 2-deoxy-D-glucose, indicating increased glucose consumption. Furthermore, glucose restriction (5.5 mM instead of 25 mM) dramatically increased the susceptibility of neuronal and glial cells to hypoxic and ischemic conditions. Glucose media concentrations below 2 mM caused selective neuronal cell death in otherwise normal culture conditions. GABAergic neurons showed a particularly high sensitivity to glucose restriction, hypoxia, and ischemia. The pattern of ischemia-induced changes in vitro showed many similarities to in vivo findings, suggesting that aggregating brain cell cultures provide a useful in vitro model to study pathogenic mechanisms related to brain ischemia.
Animals↗
Nerve growth factor (NGF) stimulation of cholinergic telencephalic neurons in aggregating cell cultures.
The addition of nerve growth factor (2.5S NGF) to serum-free aggregating cell cultures of fetal rat telencephalon greatly stimulated the developmental increase in choline acetyltransferase activity. Two other neuronal enzymes, acetylcholinesterase and glutamic acid decarboxylase, showed only slightly increased activities after NGF treatment whereas the total protein content of the cultures and the activity of 2',3'- cyclic nucleotide phosphodiesterase remained unchanged. The stimulation of choline acetyltransferase was dependent on the NGF media concentrations, showing a 50% maximum effect (120% increase) at approximately 3 ng/ml (10-10 M 2.5S NGF). NGF treatments during different culture periods showed that the cholinergic neurons remained responsive for at least 19 days. The continued treatment was the most effective; however, an initial treatment for only 5 days still caused a significant stimulation of choline acetyltransferase on day 19. The observed stimulation appeared to be specific to NGF. Univalent antibody fragments (Fab) against 2.5S NGF completely abolished the NGF-dependent increase in choline acetyltransferase activity, whereas Fab fragments of control IgG were ineffective. Furthermore, angiotensin II, added in high amounts to the cultures, showed no stimulatory effect. The present results suggest that certain populations of rat brain neurons are responsive to nerve growth factor.
Acetylcholinesterase↗
Immunocytochemical localization and developmental profile of neuron specific enolase (NSE) and non-neuronal enolase (NNE) in aggregating cell cultures of fetal rat brain.
In aggregating cultures, neuron specific enolase (NSE) was first detected biochemically at 3 days. NSE levels increased with time in aggregate cultures and at 48 days reached a level which was 33% of that found in adult rat brain in vivo. The level of non-neuronal enolase (NNE) was essentially identical in aggregate cultures and normal rat brain. Immunocytochemically, NSE(+) cells first appeared at 10 days in vitro. Their number increased until 20 days in culture and then remained constant. When the immunocytochemical localization of NSE and NNE was compared in vibratome sections of 25 day aggregates, all identifiable neurons were NSE(+), NNE(-) and glial cells were NSE(-), NNE(+). In 1 micron thick epon sections of 30 day aggregates NSE antiserum stained neuronal cytoplasm intensely. Comparison of NSE staining in 1 micron thick epon sections with the same cell in an adjacently cut thin section provided conclusive evidence that NSE(+) cells were neurons and NSE(-) cells were glia. These results demonstrate that the three-dimensional organization of aggregate cells provides an excellent environment for neuronal differentiation and also emphasize the advantages of this culture system for multidisciplinary studies of brain development.
Animals↗