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Biochemical and morphological comparison of two tumour-cell-aggregation factors from rat ascites hepatoma cells.

Two tumour-cell-aggregation factors, derived from rat ascites hepatoma cells, had different antigenicity; one was not absorbed by immunoadsorbent chromatography with anti-rat serum antibody and the other was. Their activities were both lost by digestion with trypsin, but remained unchanged by oxidation with periodate, suggesting the role of the protein portions in their molecules. The potency of the unabsorbed factor was inhibited specifically by alpha-methyl-D-mannoside or D-mannose, while that of the absorbed factor was inhibited specifically by N-acetyl-D-glucosamine, suggesting that these carbohydrates may be concerned with the respective receptor structures at the tumour-cell surface. The unabsorbed factor induced not only cell aggregation (as shown in the form of simple apposition) but also cell adhesiveness characterized by development of intermediate junctions, desmosomes and tight junctions, while the absorbed factor produced only simple apposition, suggesting their functional difference.

Animals

Production of plasminogen activator and inhibition of embryonic cell aggregation by cultured human normal and neoplastic cells.

The plasminogen activator (PA) production and the capacity to inhibit embryonic neural retina (NR) cell aggregation by human normal and neoplastic cell lines have been studied. The PA production was detected by both iodinated fibrin and casein lysis assays, and by changes in cell morphology at the presence of activated PA, using dog serum. Since the casein lysis assay and morphological changes proved to be less sensitive than 125I-fibrin lysis assay, a good correlation between these three assays could be observed provided that PA production measured by fibrinolysis exceeded 10--20%. The neoplastic cell lines exhibited the PA production to quite a large extent. The highest fibrinolytic activity (78%) was found in the case of bladder carcinoma cells T24, while the B-5GT cells from giant cell tumor of bone failed to produce any detectable amount of the PA. The cells from synovial sarcoma and both glioma lines exhibited fibrinolytic activity of about 10% and four sarcoma cell lines over the range 20--50%. Out of 13 normal cell lines tested, 7 were negative or exhibited very low fibrinolysis not exceeding 3% of total radioactivity. Four cell lines derived from kidneys, lungs, intestines, and from mixed embryonic tissues showed a marked fibrinolytic activity of about 10--37%, a slightly elevated fibrinolysis being found in embryonic lung cells LEP and cells from fetal skin tissue only at the presence of dog serum. The fibrinolysis detected in the neoplastic cloned cell populations showed considerable differences in the PA production between individual cell clones isolated from the same parental cell line. Unlike the normal fibroblastic cells B-41FB derived from bone, all neoplastic cell lines tested possess the capability to inhibit embryonic NR cell aggregation significantly. The results suggest the effect not to be dependent upon the PA production.

Animals

Embryonic tissues as elasticoviscous liquids. I. Rapid and slow shape changes in centrifuged cell aggregates.

Certain embryonic tissue masses and cell aggregates behave like deformable solids during brief experimental manipulations but like viscous liquids in long-term organ cultures. To investigate these seemingly paradoxical physical properties, we have mechanically deformed cell aggregates derived from several embryonic chick organs by centrifuging them against solid substrata. Aggregate shapes during brief centrifugation were observed directly in a microscope-centrifuge. In addition, techniques were devised for fixing cell aggregates during prolonged centrifugation. Evidence presented here shows that these fixative-injection procedures accurately preserve the prefixation shapes of living centrifuged aggregates. According to a simple viscous-liquid model for cell aggregates, cohering cells slide past one another when external forces and/or tissue surface tensions cause gradual rearrangements in aggregate conformations. In earlier experiments, 2 types of behaviour predicted from this model were confirmed for several embryonic chick tissues subjected to prolonged centrifugation. First, initially flat aggregates rounded up against the centrifugal force to adopt the same shapes that initially round aggregates reached by flattening. Second, the relative roundness of centrifuged aggregates of different tissues at shape equilibrium correlated with the relative positions that these tissues assumed when they were combined in aggregate-spreading and cell-sorting experiments. By contrast, the brief centrifugation experiments described here provide some support for a simple elastic-solid model in which aggregate shape changes are accompanied by cell deformations rather than cell redistributions. In particular, since cell migration tends to occur quite slowly, the very rapid aggregate flattening observed during the first few minutes of centrifugation presumably requires cell stretching. Moreover, since they do also round up very rapidly following brief centrifugation, these aggregates exhibit considerable elasticity that presumably reflects the swift relaxation of cell stretching as the centrifugal force is removed. Athough both elastic-solid and viscous-liquid properties can be recognized in cell aggregates, we note that, in the prolonged centrifugation experiments described here, rapid initial aggregate flattening is followed by much more gradual, continued flattening. Similarly, after prolonged centrifugation, rapid partial aggregate rounding-up is also followed by much more gradual, continued rounding-up during subsequent culture at Ig. Such rapid-then-slow shape changes contradict both simple elastic-solid and simple viscous-liquid models for cell aggregates. These bimodal shape changes are instead consistent with both compound-viscoelastic-solid and elasticoviscous-liquid models for cell aggregates, although only the latter can also account for long-term liquid-like aggregate behaviour...

Animals

[Induced cell aggregation].

Data from literature concerning cell aggregation induced by various substances are considered. The biochemical nature of cell aggregation inducers, participation of membrane receptors in this process, biochemical aspects and practical significance of the cell aggregation process are discussed.

Animals

Nevus cell aggregates associated with lymph nodes: estimated frequency and clinical significance.

Aggregates of cells resembling those of cutaneous nevi have been found in the capsules of lymph nodes. Although seemingly rare, this extraordinary lesion could conceivably occur often enough to be more than a pathological curiosity, and should be differentiated from metastatic tumor. Slides from every axillary lymph node dissection for female mammary carcinoma in the years 1964 and 1974 at Memorial Hospital were reviewed, as were slides from 100 consecutive lymph node dissections performed during 1974 in patients with malignant melanoma. Nevus cell aggregates (NCA) were associated with three of 17,504 lymph nodes (0.017%) obtained from 909 mastectomies, or 0.33% of the cases. Among the 100 lymph node dissections for malignant melanoma, NCA were found associated with three of the 2,607 lymph nodes examined (0.12%), or 3.0% of the cases studied. Since NCA occur in association with lymph nodes more often than previously thought, the possibility that they may be a potential source of malignant melanoma in patients without a demonstrable cutaneous or mucosal primary is discussed.

Axilla

Ultraviolet-induced alterations of beat rate and electrical properties of embryonic chick heart cell aggregates.

Embryonic heart cell aggregates were irradiated with ultraviolet light at wavelengths between 260 and 310 nm. Spontaneous beat rate was monitored with the aid of a closed-circuit TV camera and, in separate experiments, electrophysiological changes were assayed by intracellular recording. The characteristic response of 7-day aggregates was an increase in spontaneous beat rate to a maximum plateau level, followed by a rather abrupt cessation of beating. Intracellular recordings during irradiation showed a marked decline in the maximum rate of rise, overshoot, and repolarization phase of the action potential, and a significant change in threshold toward zero. The action spectrum for the termination of beating peaked between 290 and 295 nm; it fell off sharply at longer wavelengths and more slowly at shorter wavelengths. The maximum increase in beat rate was increasingly greater for shorter wavelengths and exhibited no peak in the wavelength range investigated. The sensitivity of aggregates to 295-nm light, as measured by the inverse of irradiation time required to terminate beating, decreased with increasing aggregate size and external potassium concentration, was relatively independent of temperature, and increased with embryonic age. The ultraviolet-induced increase in beat rate and termination of beating are attributed to separate complementary processes, a depolarization of the membrane, and a decline in "fast" sodium conductance.

Action Potentials

Evidence for a pertussis toxin-sensitive signalling pathway in the dual action of angiotensin II on growth hormone release in pituitary cell aggregates.

In anterior pituitary cell aggregates cultured in the presence of the glucocorticoid dexamethasone (DEX) angiotensin II (AII) had a dual effect on growth hormone (GH) release. The peptide stimulated the release in aggregates from 2-week-old rats, whereas the peptide had an inhibitory effect in cultures from adult rats. Treatment of aggregates from adult rats with pertussis toxin (PT) reversed the inhibitory effect of AII on GH release in a stimulatory effect; PT treatment of aggregates from 18- to 20-day-old rats significantly enhanced the stimulation of GH release by AII. The effect of PT was seen only when DEX was added to the culture medium. The present data suggest that the glucocorticoid-dependent stimulus-effect coupling of AII on GH release involves both a stimulatory and an inhibitory component, the latter being abolished by PT, and that the stimulatory component predominates during immature life while the inhibitory one during adult life.

Age Factors

Do nonionic contrast media increase red cell aggregation and clot formation?

Red cell aggregate formation was studied both macroscopically and microscopically in blood mixed with a contrast medium, either ioxaglate or iohexol. Whole blood was layered on top of the contrast medium, and after being shaken, blood cells were separated from the medium by centrifugation. The kinetics of red cell aggregate formation were measured by a transparent rheoscope and a photoaggregometer at different shear rates. Coagulation studies were conducted on citrated platelet-rich plasma mixed with contrast media. Platelet shapes were observed with phase contrast optics, and coagulation times were measured. It was demonstrated that the increased red cell aggregation, which occurred during the initial contact between blood and the contrast medium, dispersed after being submitted to shear force and did not re-form in the remaining plasma. Since this phenomenon is associated with high concentrations of contrast media in nonflowing blood, the high shear rate in arteries and arterioles make it unimportant in the in vivo situation. Within highly concentrated contrast media solutions, blood clot formation was not inducible, and the risk of thrombus formation in fresh blood drawn into contrast medium-filled syringes appears minimal.

Blood Coagulation

Production and turnover of cAMP signals by prestalk and prespore cells in Dictyostelium discoideum cell aggregates.

Dictyostelium discoideum prestalk cells and prespore cells from migrating slugs and culminating cell aggregates were isolated by Percoll density centrifugation. Several activities relevant to the generation, detection, and turnover of extracellular cyclic AMP (cAMP) signals were determined. It was found that: the two cell types have the same basal adenylate cyclase activity; prespore cells and prestalk cells are able to relay the extracellular cAMP signal equally well; intact prestalk cells show a threefold higher cAMP phosphodiesterase activity on the cell surface than prespore cells, whereas their cytosolic activity is the same; intact prestalk cells bind three to four times more cAMP than prespore cells; no large differences in cAMP metabolism and detection were observed between cells derived from migrating slugs and culminating aggregates. The results are discussed in relation to the possible morphogenetic role of extracellular cAMP in Dictyostelium cell aggregates. On the basis of the properties of the isolated cells we assume that a gradient of extracellular cAMP exists in Dictyostelium aggregates. This gradient appears to be involved in the formation and stabilization of the prestalk-prespore cell pattern.

3',5'-Cyclic-AMP Phosphodiesterases

Characterization of tumour cell aggregation promoting factor from rat ascites hepatoma cells: Separation of two factors with different antigenic property.

The previously described glycoprotein that promotes tumour cell aggregation, derived from rat ascites hepatoma cells and capable of partial purification by chromatography, was found to be a mixture of 2 factors with different antigenic property. One was not absorbed by immunoadsorbent chromatography with anti-rat serum antibody and the other was. The action of the unabsorbed factor was clearly more potent than that of the absorbed factor. Both the factors were found in the serum of tumour bearing rats and the action of the unabsorbed factor was also more potent than that of the absorbed factor; its amount increased with time after i.p. inoculation of the cells. The serum of healthy rats contained the absorbed factor but not the unabsorbed factor. It was thus assumed that the unabsorbed factor was associated with the hepatoma cell surface itself and released into the serum, while the absorbed factor was associated with serum protein coating the cell.

Animals

Red cell aggregation and the echogenicity of whole blood.

To study the relationship between red cell aggregation and whole blood echogenicity, red cell aggregation was quantitated by a photometric method, whole blood echogenicity was quantitated by videodensitometry and sedimentation rate was quantitated by a modified Westergren method. Changes in red cell aggregation were produced by alterations in the hematocrit. The results showed that red cell aggregation increased in a linear fashion with increases in hematocrit. The sedimentation rate decreased in a linear manner with increases in hematocrit. Whole blood echogenicity showed a biphasic response, with an initial increase in echogenicity, peaking at hematocrits varying from 14-24% and decreasing thereafter. Over the physiologic range of hematocrits, an increase in the formation of red cell aggregates is associated with a decrease in the echogenicity of whole blood. Thus, red cell aggregates were not visible using our ultrasound equipment at physiologic hematocrits, and the echo contrast in blood under our experimental conditions at these hematocrits must represent either plasma spaces, platelet aggregates or possibly white cell aggregates. The association between spontaneous contrast and a propensity for thromboembolism imply that platelet aggregates are the most likely origin of in vivo echo contrast in flowing blood.

Animals

Role of hydrogen bonding in red cell aggregation.

The role of hydrogen bonding in red cell aggregation induced by dextran was studied with the use of urea, an inhibitor for hydrogen bonding. In order to avoid hemolysis of red cells by the high concentration of urea, the studies were performed on human red cells hardened in glutaraldehyde. The degree of red cell aggregation at Hct = 45% was estimated by the use of a coaxial cylinder viscometer. The viscometric aggregation index (VAI) was calculated from viscosity values at shear rates of 52 sec-1 (eta H) and 0.05 sec-1 (eta L); VAI = (eta L - eta H)/eta H. Red cells with surface charge intact and with charge removal by neuraminidase treatment were studied. Urea at high concentrations, e.g., 6 M, significantly inhibited red cell aggregation induced by dextran. These findings indicate that hydrogen bonding plays an important role in dextran-induced red cell aggregation. An understanding of the nature of the forces involved in red cell aggregation serves to establish the physicochemical principles of cell-to-cell interactions induced by macromolecules.

Blood Viscosity

Importance of cell-aggregation during induction of neural differentiation in PCC-7 embryonal carcinoma cells.

The importance of cell-aggregation during retinoic acid-induced neural differentiation of embryonal carcinoma cells was studied on the PCC-7 cell line. These cells were chosen as they display low tendency for spontaneous aggregation, and they develop preferentially to neurons upon induced in vitro differentiation. Forced aggregation of these cells, in the absence of retinoic acid, did not result in development of neuron- or glial-like cells. Application of retinoic acid prior to or after the cell-aggregation did not result in neural tissue-like differentiation, either. Irreversible induction of neural development was achieved if cell-aggregation and retinonic acid acted simultaneously, and for a period longer than 48 h. Retinoic acid, on the other hand, was found to be toxic on non-aggregated PCC-7 cells. Our data suggest that cell to cell contacts alter the response of these cells to retinoic acid, and their close apposition is a prerequisite for the retinoic acid-induced neural differentiation.

Animals

Purification and characterization of the retina-specific cell-aggregating factor.

The tissue-specific, cell-aggregating component of embryonic neural retina cells was purified from the retina cell-aggregating factor and characterized as a glycoprotein. Its molecular weight in solution is in the range of 50,000, and it contains 10-15% carbohydrate. The amino-acid and carbohydrate compositions have been determined. The glycoprotein is produced by embryonic neural retina cells in primary monolayered cultures and is released into the medium. Its tissue-specific, cell-aggregating effect requires integrity of the polypeptide portion, but not of the carbohydrate portion. We suggest that the isolated molecule is a specific determinant of the embryonic retina cell-surface and that it is involved in mediating self-recognition and selective adhesiveness of these cells.

Amino Acids

A monoclonal antibody that induces T cell aggregation reacts with vascular endothelial cells and placental trophoblasts.

We have found that a mouse monoclonal antibody (alpha Leu-13) to a 16 kilodalton human lymphocyte surface antigen reacts with vascular endothelial cells as determined by immunoperoxidase staining of frozen tissue sections. In earlier studies, alpha Leu-13 was found to induce purified T cells to aggregate when added to cultures in nanogram concentrations. In the studies reported here, alpha Leu-13 stained vascular endothelial cells of arteries, capillaries, and veins in all organs examined from adults. It also reacted weakly with epithelial cells of proximal tubules of the kidney and with nonkeratinized basal epithelial cells of the cervix and esophagus. When a panel of tissues from a 14-wk-old fetus was examined, alpha Leu-13 was not found to react with endothelial cells of any specimen. However, it did stain medullary thymocytes and placental trophoblasts of this fetus. The implications of these findings to the possible function of the Leu-13 antigen in immune ontogeny are discussed.

Antibodies, Monoclonal

Patterning in hydra cell aggregates without the sorting of cells from different axial origins.

Aggregates of Hydra cells were studied to find out how the primary centers that form new heads are generated in a system of cells in which the original pattern has been destroyed. Since cells that originate near the heads (apical cells) temporarily maintain a high level of head activation potential during aggregate formation and may contribute to pattern formation, their distribution in the aggregates was investigated. The mutual distances between labeled epithelial cells were followed by vitally staining apical cells with DAPI. The distribution was random during early regeneration stages (3, 6, 24 hr). These results show that epithelial cells originating from apical regions do not sort. That is, dynamic cell movement to generate rudiments of new heads is not necessary for head formation in aggregates. A possible explanation of the mechanism is discussed.

Animals