Enucleation eliminates a differentiation-specific surface marker from normal and leukemic murine erythroid cells

Mammalian erythropoiesis includes a step in which the nucleus is extruded through the cell membrane. We have investigated the relationship between concanavalinA (conA) plasma membrane receptors, which are known to leave the incipient reticulocyte during enucleation, and regions of the plasma membrane which bind merocyanine 540, a differentiation-specific marker of hematopoietic cells. The distribution of these two fluorescent probes was examined on living cells from the spleens of neonatal mice and on erythroleukemia cells induced to enucleate in culture. In both cases, the region of the membrane extruded with the nucleus preferentially binds conA and merocyanine 540, whereas the plasma membrane which is left behind retains the capacity to bind another lectin, wheat germ agglutinin (WGA). The implications of these findings are discussed with respect to the mechanism by which markers are eliminated from the erythrocyte cell surface.     

Injected mitotic extracts induce condensation of interphase chromatin

Although extracts from mitotic cells have been shown to induce chromosome condensation when injected into amphibian oocytes, they have not as yet been shown to induce this response in somatic interphase cells. In the experiments reported here, when mitotic extracts were injected into syncytial frog embryos, whose somatic nuclei were arrested in interphase, chromosome condensation was observed. The inability of interphase extracts, injected at similar concentrations, to induce this event demonstrates the cell cycle-specific accumulation of the factors responsible.     

Adhesive and migratory behavior of normal and sulfate-deficient sea urchin cells in vitro

Dissociated cells from different stage embryos of the sea urchin Lytechinus pictus were compared in their adhesion to various substrates. Micromeres from 16-cell stage embryos bind to tissue culture and Petri dishes but not to Petri dishes coated with human plasma fibronectin. Other cell types did not adhere to any of the substrates tested. By hatched blastula stage, about 28% of the cells adhered to fibronectin as well as to tissue culture dishes. By the mesenchyme blastula stage, there was a further increase in the proportion of cells adhering to these substrates. At no stage did cells adhere to native rat tail collagen. Primary mesenchymal cells were isolated by their selective adhesion to tissue culture dishes in the presence of horse serum. These cells were then examined for their migratory capacity. Cell spreading and migration followed adhesion and occurred on fibronectin but not on the other substrates tested. Based on analysis of video tapes, greater than 60% of these cells moved faster than 1 micron/min. On the other hand, cells from sulfate-deprived embryos, in which primary mesenchyme migration is blocked in situ, failed to spread and migrated little on the same substratum. This defect was reversed by a 6 h pretreatment of the cells in normal sea water. Thus, the in vitro migratory behavior parallels that observed in vivo. These results support the hypothesis that the primary mesenchymal cells produce a sulfate-dependent component that is required for cell spreading and migration.     

The cellular oncogenes c-myc, c-myb and c-erb are transcribed in defined types of avian hematopoietic cells

The possible role of normal chicken cellular sequences c-erb, c-myb and c-myc, together referred to as c-onc genes and related to the oncogenes of defective avian acute leukemia retroviruses (DLVs), was investigated by determining the accumulation of c-onc RNA in different avian cells an cell lines. Levels of c-myc and in some instances c-myb RNA are elevated in immature hematopoietic cells or cell lines from various lineages but more mature hematopoietic cells, as well as non-hematopoietic cells, contain only low levels. In contrast, the level of c-erb RNA is generally low, but high in a small number of normal bone marrow cells. The results indicate that the cellular homologues of the viral oncogenes are differentially expressed during hematopoiesis. They also indicate that the hypothesis that DLV target cells express their homologous c-onc genes might hold for c-erb, but is not valid in its simple form for c-myc and c-myb.     

Phagocytosis of lectin-coated beads by dystrophic and normal retinal pigment epithelium

In the dystrophic pigmented Royal College of Surgeons (RCS) rat, the retinal pigment epithelium (RPE) has a diminished capacity to phagocytose shed photoreceptor outer segments (ROS). An alteration in phagocytic recognition or ligand-receptor interactions between the RPE and ROS's could contribute to this defect. To this end, we have examined whether or not RPE lectin receptors are implicated in phagocytosis in the normal and dystrophic rat RPE by comparing differences in phagocytic uptake of lectin-coated beads. To test this, the following lectins were bound either indirectly to sugar-coated latex beads or directly to activated beads: Concanavalin A (conA), specific for mannose; Ulex europeus (ULEX), specific for fucose; Lens culinaris (LcH), specific for mannose; and wheat germ agglutinin (WGA), specific for N-acetyl glucosamine and sialic acid. The distribution of the lectin binding around beads was visualized and confirmed using lectin-Ferritin conjugates. Lectin-coated beads were fed to normal and dystrophic pigmented RPE tissue explants to determine differences in phagocytic uptake. We found that whether beads were directly or indirectly coated, similar results were obtained, but that there were differences in uptake of two types of lectin-coated beads by dystrophic as compared with normal animals. The dystrophic RPE phagocytosed greater numbers of conA-mannose beads (6.9/cell) than the normal RPE (3.6/cell). LcH-mannose beads were also phagocytosed by dystrophic (2.7/cell) but not by the normal (0/cell). A similar number of ULEX-fucose beads were taken up by dystrophic (3.8/cell) and normal (3.4/cell) RPE and neither took up WGA-N-acetyl glucosamine beads (0/cell). These results showing that the dystrophic RPE takes up greater numbers of conA and LcH-coated beads than the normal RPE suggest that a ligand-receptor interaction involving mannose may contribute to this difference in phagocytic uptake.     

The effect of monensin on cell aggregation of normal and dystrophic human skin fibroblasts

Measurements of aggregation kinetics using couette viscometry show that freshly trypsinized skin fibroblasts from patients with Duchenne muscular dystrophy have values of intercellular adhesiveness approx. 40% those of normal cells. If cells are allowed to recover from the effects of trypsinization (by incubation for 2 h at 37 degrees C in serum-containing medium) the intercellular adhesiveness of both cell types increases, and normal and Duchenne cells aggregate to the same extent. Exposure to the ionophore monensin during the recovery phase leads to suppression of recovery in both cell types, and this effect of the drug is greater in Duchenne fibroblasts. These results are discussed in relation to other data on the reported differential effects of trypsin and monensin on normal and Duchenne fibroblasts.     

Fucoidin inhibits attachment of guinea pig spermatozoa to the zona pellucida through binding to the inner acrosomal membrane and equatorial domains

Our previous study has shown that fucoidin, an algal heteropolysaccharide, is a potent inhibitor of sperm-zona binding in the guinea pig, hamster and human. To visualize the surface site of fucoidin binding, a biotinated derivative (B-Fuc) of the native fucoidin was prepared. B-Fuc retained the inhibitory activity and was used in conjunction with FITC-avidin to localize its binding sites on guinea pig spermatozoa using fluorescence microscopy. In living acrosome-reacted spermatozoa, B-Fuc bound predominantly to the inner acrosomal membrane and equatorial segment domains. The binding was effectively competed by a 10-fold excess of native fucoidin, but not by a 10-fold excess of heparin or a 20-fold excess of biotinated normal rabbit serum IgG. B-Fuc binding patterns on dead spermatozoa were quite different from that of living spermatozoa. The post-acrosomal region, rather than the inner acrosomal membrane and equatorial domains, was intensely labeled. This indicates the importance of using living cells in assessing true surface binding sites whenever possible. We conclude that the inner acrosomal membrane and/or equatorial domains are critical for zona binding in the guinea pig.     

Proliferation and differentiation of chick skeletal muscle cells cultured in a chemically defined medium

By using the technique of nuclear transplantation in Paramecium [1], amicronucleate and renucleate clones were prepared in P. caudatum. The major differences between amicronucleate and micronucleate cells in the vegetative stage are elongation of cell cycle time, decrease in food vacuole formation, and shortening of the buccal cavity in the amicronucleate cells. These characteristics of amicronucleate cells are closely related with the absence of micronucleus, because all of these abnormalities were cured when the micronucleus was transplanted again into the amicronucleate. It is evident that the germinal micronucleus plays an important role not only during the sexual cycle but also in vegetative growth. Elongation of the cell cycle time in amicronucleates was also observed in P. bursaria and P. jenningsi.     

The effects of retinoic acid on [14C]acetate incorporation into lipids of normal and transformed hamster fibroblasts

This study examined the effects of retinoic acid (RA) on [14C]acetate incorporation and fatty acid composition of hamster embryo fibroblasts (HEF) and two cell lines derived from the same inbred strain but transformed by herpes simplex-2 virus (HSV) or polyoma virus (HFT). Cells were exposed to all trans RA, or dimethylsulfoxide (DMSO), the vehicle for RA, and the lipids labeled with [14C]acetate. Lipids were extracted from the cells, separated by paper chromatography, located by autoradiography, and acetate incorporation determined by liquid scintillation spectrometry. The distribution of fatty acids in total cell lipids was examined by gas chromatography. HEF cells incorporated more acetate into cholesterol than either transformed cell type. The HFT line incorporated more acetate into triglycerides and less into total phospholipids than either the HSV line or the HEF line. RA caused a significant decrease in incorporation of acetate into cholesterol and sphingomyelin in all three cell lines. HEF and HSV cells had decreased incorporation into phosphatidyl inositol-phosphatidyl serine and increased incorporation into triglycerides, changes not evident in the HFT cell. The control fatty acid profiles of the HEF and HSV cells were similar, while the HFT cells had a larger proportion of C16:0 and 18:1 fatty acids. Following treatment with RA all three cell types showed an increase in palmitic and a decrease in oleic acids. The three related cell types showed different [14C]acetate labeling patterns which did not respond uniformly to RA. On the other hand, exposure elicited some like responses in all cell types.     

The synthesis and intracellular localization of adenovirus hexon protein studied by microinjection of mRNA into human cells

Polyadenylated RNA isolated 18 h after infection of HeLa cells with adenovirus type 2 was both translated in vitro and microinjected into the cytoplasm of human transformed amnion cells. The hexon polypeptide could be specifically immunoprecipitated from the products of cell-free translation with a rabbit-anti-hexon serum. The same serum when used in immunofluorescence assays of microinjected cells revealed hexon protein synthesized 6 h after microinjection. The intensity of the staining persisted up to 16 h after injection of messenger RNA (mRNA). Newly-synthesized hexon protein was characteristically located mainly in the nucleus. Essentially the same results were obtained when normal amnion cells were microinjected.     

Pyruvate regulation of growth and differentiation in primary cultures of rat tracheal epithelial cells

These studies examined the effect of exogenous pyruvate on the growth and differentiation of primary cell cultures of rat tracheal epithelial cells. The cell cultures were derived from outgrowths of tracheal explants, and require pyruvate for survival and growth in the presence of 10% FBS. In pyruvate-supplemented (2 mM) medium, the number of cells attached to the dish increased rapidly, while exfoliation of cells into the medium as well as formation of cornified envelopes were relatively low. The growth response to pyruvate was concentration-dependent in these cell cultures. In the absence of pyruvate, the extent of terminal differentiation to keratinization gradually increased. This was characterized by a cessation of growth after one week, and an increase in exfoliation until all cells had sloughed from the dish. Accompanying these changes was a marked increase in the formation of cornified envelopes. Cells undergoing DNA synthesis were present throughout 2 weeks of culture in pyruvate-deprived medium, even as the total number of cells was diminishing. Several compounds, including other 2-oxocarboxylic acids, were ineffective growth substitutes for pyruvate. These results indicate that the requirement for pyruvate is quite stringent in these cultures and that one way pyruvate promotes the growth of tracheal epithelial cells is by inhibiting terminal differentiation.     

Contribution of alpha-D-galactopyranosyl end groups to attachment of highly and low metastatic murine fibrosarcoma cells to various substrates

There are much greater numbers of cell surface terminal, non-reducing alpha-D-galactorpyranosyl groups in highly malignant (metastatic) cells than are found in low malignant cells derived from the same murine fibrosarcoma. We have examined the contribution of these residues to attachment of the cells to various collagens and to plastic. Removal of these carbohydrate groups with alpha-galactosidase or blocking them with lectins from Griffonia simplicifolia seeds or with anti-blood group B antiserum all dramatically inhibited the attachment of both the highly malignant and the low malignant cells. Following removal with the enzyme, the alpha-D-galactopyranosyl end groups were rapidly resynthesized. This resynthesis was inhibited by tunicamycin, an inhibitor of de novo glycoprotein synthesis. This antibiotic also impaired cell attachment and, when used in addition to treatment with alpha-galactosidase, it inhibited cell attachment more than did treatment with the enzyme alone. The effects of all treatments on cell attachment were greater for the highly malignant than for the low malignant cells. With the latter cells, inhibition by lectin was seen only in the absence of serum, whereas the adhesion of highly malignant cells was affected in both the presence and the absence of serum. On their surface membrane the highly malignant cells express much more than do the low malignant cells of a glycoprotein that cross-reacts immunologically with laminin. The basement membrane glycoprotein laminin promotes cell attachment to collagen, and both glycoproteins contain terminal, non-reducing alpha-D-galactopyranosyl groups. Attachment of cells is a requirement for the formation of a metastasis, and thus the laminin-like molecule and the alpha-D-galactopyranosyl end groups (whether on the laminin-related moiety or on other cell surface molecules) may both be important for expression of the most malignant phenotype.     

Glucose metabolite patterns as markers of functional differentiation in freshly isolated and cultured mouse mammary epithelial cells

In the mammary gland of non-ruminant animals, glucose is utilized in a characteristic and unique way during lactation [11]. By measuring the incorporation of glucose carbon from [U-¹⁴C]glucose into intermediary metabolites and metabolic products in mammary epithelial cells from virgin, pregnant, and lactating mice, we demonstrate that glucose metabolite patterns can be used to recognize stages of differentiated function. For these cells, the rates of synthesis of glycogen and lactose, the ratio of lactate to alanine, and the ratio of citrate to malate are important parameters in identifying the degree of expression of differentiation. We further show that these patterns can be used as markers to determine the differentiated state of cultured mammary epithelial cells. Cells maintained on plastic substrates lose their distinctive glucose metabolite patterns while those on floating collagen gels do not. Cells isolated from pregnant mice and cultured on collagen gels have a pattern similar to that of their freshly isolated counterparts. When isolated from lactating mice, the metabolite patterns of cells cultured on collagen gels are different from that of the cells of origin, and resembles that of freshly isolated cells from pregnant mice. Our findings suggest that the floating collagen gels under the culture conditions used in these experiments provide an environment for the functional expression of the pregnant state, while additional factors are needed for the expression of the lactating state.     

Changes in cell-substratum adhesion and nuclear-cytoskeletal anchorage during cytodifferentiation of BeWo choriocarcinoma cells

Changes in the substratum anchorage of cells and nuclei were examined during methotrexate (MTX)-induced cytodifferentiation of BeWo human choriocarcinoma cells. During this process cytotrophoblast-like cells (CTLs) transform into giant mono- and multinucleated syncytiotrophoblast-like cells (STLs). Cells treated with MTX for 24 h exhibited significantly faster rates of substratum detachment by EDTA, trypsin-EDTA, EDTA-glycine, and DMSO than did uninduced controls. The decrease in cell-substratum adhesiveness occurred prior to the onset of morphological transformation. By 48 h, when morphological transformation was first observed, there had occurred a marked change in nuclear-cytoskeletal anchorage to the substratum, as evidenced by a difference in sensitivity of Triton-extracted STL and CTL monolayers to detachment by KI. STL monolayers were completely detached within 5 min of exposure to 0.3 M KI, while CTL monolayers remained firmly attached to the substratum for at least 3 h. KI-extracted residues were examined by electron microscopy and found to consist of nuclear shells attached to intermediate filaments. When cytoskeletal residues and KI-extracted proteins of STL and CTL cells were compared by two-dimensional polyacrylamide gel electrophoresis (PAGE), qualitative and quantitative differences were seen in a number of minor components. Thus the sensitivity of STL nuclear-cytoskeletal monolayers to removal by KI, an effective actin depolymerizing agent, may involve changes in the organization, stability, or interactions of actin with other components of the cytoskeletal framework.     

Synchronous exocytosis in Paramecium cells. III. Rearrangement of membranes and membrane-associated structural elements after exocytosis performance

Aminoethyldextran (AED) was used to trigger the synchronous release of trichocysts from Paramecium tetraurelia cells (see [8]) by a mechanism involving exocytotic membrane fusion and resealing (see [5]). Ultrastructural changes were analyzed by quantitative evaluation of ultrathin sections. In resting cells the percentage of potential trichocyst-docking sites which are actually occupied by a trichocyst was 58%; 36% of potential docking sites contained ghosts and 6% a "plug" of electron-dense material. We derived from our data that paramecia would discharge permanently and spontaneously trichocysts (without AED) at a rate of 2-3 per min (which we then also verified by counting the spontaneous release rate) and that this value is equivalent to the docking rate. For the synchronous expulsion of trichocysts in response to AED we had determined that the degree of synchrony is more than a hundred times better than in most other systems (see [8]). We have determined the half-lives (HL) for different events involved in exocytosis and re-docking as follows: approximately 3 sec for trichocyst discharge, approximately 3 sec for the formation of ghosts, 8 min for the clearing of ghosts from the cell surface, 4 min for the formation of "plugs". Trichocysts are docked with a HL of 40 min and "plugs" (considered as receptor-type structures for trichocyst docking) disappear with a concomitant HL of 50 min. Evidently the clearing of ghosts allows for re-formation of "plugs" but the respective HL values signal that "plugs" may also be formed anew. The relatively slow decline of the percentage of "plugs" (after their azimuth 15 min after AED triggering) may also indicate the synthesis of new docking sites. After a period of over approximately 3 h following AED triggering, the original situation is roughly re-established and maintained over the whole period of population growth analyzed.     

Binding uptake and degradation of antithrombin III X protease complexes by cultured corneal endothelial cells

Interaction of 125I-labeled human antithrombin III (125I-AT III) X protease complexes with bovine corneal endothelial cells has been studied in tissue culture. 125I-AT III does not bind to endothelial cells, but its complexes with either thrombin or trypsin bind specifically to the cultures. The binding of 125I-AT III X protease complexes is not via the moiety of the free antithrombin III (AT III) or the free protease, since neither AT III nor thrombin compete on the binding of 125I-AT III X thrombin complexes. Only unlabeled AT III X thrombin complexes compete on the binding of the iodinated ligand. 125I-AT III X trypsin complexes bind with a KD of 1.4 X 10(-7) M to high affinity-binding sites present on the cell surface of corneal endothelial cells. Saturation of binding to the cell surface is observed at a concentration of 2.5 X 10(-7) M 125I-AT III X trypsin complexes and the number of binding sites per cell is about 4 X 10(4). The cell surface binding reaches a maximum by 15 min and then decreases with time. The cells, when incubated at 37 degrees C, appear to internalize the bound complexes by adsorptive endocytosis which proceeds at a rate of 0.5-0.8 pmole/1 X 10(6) cells/h. The internalization process of 125I-AT III X protease complexes is saturated at a concentration of 2.5 X 10(-7) M. Since the cells release 125I-labeled material into the extracellular media which cannot be precipitated by trichloroacetic acid (TCA), it probably represents degradation of 125I-AT III X protease complexes into small fragments at a linear rate of about 0.5 pmole/1 X 10(6) cells/h. The described process of AT III X protease complexes binding, internalization and subsequent degradation by corneal endothelial cells may represent a clearing mechanism for extracellular AT III X protease complexes formed under pathological conditions.     

Protein synthesis in lactating guinea-pig mammary tissue perfused in vitro. II. Biogenesis of milk-fat-globule membrane proteins

Guinea-pig mammary tissue was perfused in vitro, radiolabelled with [35S]methionine and intracellular protein precursors of the milk-fat-globule membrane (FGM) recovered by immunoabsorption techniques. Labelled xanthine oxidase was solely detected in post-microsomal supernatants and butyrophilin in carbonate-washed membranes. A major glycoprotein (Gp 55), was initially present in a membrane-bound form, but after longer perfusion times a fraction of this protein was recovered in the post-microsomal supernatant. These results are discussed with reference to formation of the apically-derived FGM.     

Control in previous and present generations of preparation for entry into S phase and the relationship to resting state in 3Y1 rat fibroblastic cells

In both the presence and absence of serum, 3Y1 rat fibroblastic cells synchronized at early S phase by aphidicolin entered M phase 6 h after removal of aphidicolin. However, in the second generation their entry into S phase in the presence of serum was delayed due to the deprivation of serum in the first generation. A similar delaying effect in the second generation was observed when the resting cells were stimulated by serum and then deprived of serum during a period of 8 h preceding mitosis. In both cases, the interval between mitosis and entry into S phase in the second generation was almost equal to that required for the resting cells to enter S phase when stimulated by serum. A similar delaying effect was also observed when the cells, synchronized at early S phase, were kept in suspension culture in the presence of serum for a period in the first generation. Results of a similar type of experiments using various combinations of growth factors showed that, when the G1 period in the second generation was shortened by exposure to growth factors in the first generation, and when the resting cells were stimulated to enter S phase, the same combination of growth factors was required. These and previous results suggest that the preparation for entry into S phase is controlled in both previous and present generations of 3Y1 cells.     

Effects of 12-O-tetradecanoylphorbol-13-acetate (TPA), retinoic acid and diazepam on intercellular communication in a monolayer of rat liver epithelial cells

We have studied the influence of the phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA), the vitamin A derivative retinoic acid and the benzodiazepine diazepam on intercellular communication via established gap junctions in a monolayer of rat liver epithelial cells (RLB) at various times of incubation. Intercellular communication was measured as the transfer of [3H]hypoxanthine-derived nucleotides between RLB hypoxanthine guanine phosphoribosyl transferase+ (HPRT+) and RLB HPRT- cells. TPA only showed transient inhibition of metabolic cooperation: after 4 h of treatment, intercellular communication was reduced to about 40% of the control and longer treatments showed progressively less effect until 24 h of treatment, when no difference was seen between TPA-treated and control preparations. Retinoic acid was a more effective inhibitor: both 3 X 10(-6) M applied for 24 h and 10(-4) M applied for 6.5 h, caused a 50% inhibition of label transfer. The junctional communication could only be blocked at very high concentrations (5 X 10(-4) M) in short-exposure experiments, but this is possibly a consequence of non-specific effects on the cell membrane. When the incubation time was 24 h, a considerable portion of the gap junctions appeared to persist in the 'open' state. Diazepam showed no significant inhibitory effect in the experiments performed.     

Self-regulation of membrane fluidity The effect of saturated normal and methoxy fatty acid supplementation on Tetrahymena membrane physical properties and lipid composition

Tetrahymena cells elongated and desaturated massive supplements of palmitic or lauric acid at nearly twice the rates employed by unfed cells, thereby maintaining constant the physical properties of their membrane lipids. However, when a mixture of the 9- and 10-monomethoxy derivatives of stearic acid was administered, these compounds were incorporated without further metabolism. The marked fluidizing effect of the phospholipid-bound methoxy-fatty acids elicited an immediate reduction in fatty acid desaturase activity, the pattern of change very similar to that induced by supplements of polyunsaturated fatty acids. The modulation of fatty acid desaturase activity by methoxy-acids clearly seems to be governed by embrane fluidity rather than by some form of end product inhibition of the type which might have been postulated to explain the similar effect caused by polyunsaturated fatty acids.