Selective inhibition of calcium-stimulated cation-induced pinocytosis by starvation and inhibitors of protein synthesis in Amoeba proteus

The capacity of Amoeba proteus to form pinocytotic channels after pretreatment with either puromycin, cycloheximide, emetine or a long period of starvation was studied. The effect on pinocytosis of the three inhibitors of protein synthesis was similar. They preferentially affected pinocytosis induced by Na+ with little effect on K+-induced pinocytosis. In Ca2+-deficient media, Na+-induced pinocytosis was inhibited, while the addition of Ca2+ restored channel formation. The degree of inhibition of Na+-induced pinocytosis was influenced by the concentration of Ca2+ in the inducing solution. Selective Ca2+-reversible inhibition of Na+-induced pinocytosis also occurred after starvation or treatment with a proteolytic enzyme, subtilisin. The membrane potential in starved or emetine-treated cells in culture medium was normal and their depolarising response to inducers was not diminished in solutions containing Na+. The resting input resistance of these cells was higher than in normal amoebae, but no significant difference in electrical parameters was observed after pinocytosis was induced. It is suggested that starvation, inhibition of protein synthesis, and enzyme digestion deplete the membrane of structures which are necessary for normal Ca2+ functions during induction of pinocytosis by Na+-like inducers.     

Reversible pinocytosis of horseradish peroxidase in lymphoid cells

A detailed study of fluid phase endocytosis of horseradish peroxidase (HRP) in rat lymph node cells (LNC) is presented in this paper. Preliminary experiments have shown that HRP was internalized by non-receptor-mediated endocytosis and interacted minimally or not at all with plasma membrane of LNC, and can then be considered as a true fluid phase marker for these cells. Kinetics of uptake of HRP was found not to be linear with incubation time at 37 degrees C and deviation from linearity can be attributed to constant exocytosis of HRP. The kinetics of exocytosis cannot be described by a single exponential process. Rather, a minimum of two exponentials is required to account for exocytosis. This suggests that at least two intracellular compartments are involved in this process. The first turns over very rapidly with a t 1/2 release of about 3 min and is saturated after 10 min of exposure with HRP. The second, which turns over very slowly, is characterized by a t 1/2 release of about 500 min and accounts for the intracellular accumulation of HRP. Similar biphasic kinetics of exocytosis were observed with unfractionated LNC, with T lymphocyte-enriched LNC and with lymphocytes purified according to their density. This suggests that most, if not all, LNC are able to release HRP and that each cell type is endowed with the two intracellular compartments. Kinetics of uptake of HRP in these two compartments indicated that they are probably filled by two endocytic pathways, at least partially independent. Taken together, these results seem to indicate that a rapid membrane recycling occurs in lymphocytes. Furthermore, the weak base ammonium chloride and the carboxylic ionophore monensin were shown in our study to inhibit fluid phase endocytosis of HRP. The inhibition was time-dependent and required a preincubation of the cells with the drugs to be observed. Our results suggest that a perturbation of the vesicular traffic or a sequestration of membranes involved in HRP uptake is induced by these drugs. Under these conditions the release of cell-associated HRP was also reduced and to the same extent as the inhibition of uptake. Distribution of HRP between the two compartments and the t 1/2 release of HRP from either compartment were not perturbed. Taken together these results seem to indicate that exocytosis is not specifically affected by these drugs. Inhibition of uptake in drug-treated cells could result from a general decrease of membrane recycling or to the formation of smaller pinocytic vesicles with a different surface to volume ratio.     

Thymidine triphosphate synthesis in senescent WI38 cells : Relationship to loss of replicative capacity

The effect of in vitro age on thymidine triphosphate (TTP) synthesis was assessed in WI38 cultures according to the following measurements: (1) thymidine kinase activity of broken cell preparations; (2) in situ incorporation of [3H]thymidine into acid-soluble material; and (3) total intracellular TTP content as determined by an enzymatic assay. All three parameters were maximal in exponentially proliferating populations and minimal in quiescent monolayers; no significant differences between young and old cultures were observed despite the reduced replicative capacity of the latter. The addition of serum to density-arrested cultures induced both TTP synthesis and DNA replication after a lag of approx. 12 h; although a greater percentage of young cells initiated replication as compared with old, pool sizes expanded to a similar extent in both populations. Pool expansion did not require entry into S phase; the pool sizes of control and cytosyl arabinoside-treated cultures were comparable. These findings suggest that senescent cells retain the ability to synthesize TTP, even though they are incapable of replicating DNA. Because TTP synthesis is a cell cycle-dependent event that normally begins in late G1, senescent cells might be blocked in the latter portion of the prereplicative phase and not in G0 as are quiescent cells.     

Two sites for calcium action in compaction of the mouse embryo

Compact mouse morulae were decompacted in calcium-free medium and allowed to recompact in standard embryo culture medium. When the recompaction medium contained trifluoperazine (TFP)(0.05 mM), an inhibitor of the calcium-dependent protein calmodulin, the embryos failed to recompact. This effect could not be overcome by either db-cAMP (1.0 mM) or theophylline (0.75 mg/ml). When the recompaction medium contained less than standard calcium (0.085 or 0.17 mM) the embryos recompacted at a slower rate than in control medium (1.7 mM). The calcium ionophore A23187, at concentrations up to 1.5 X 10(-3) mM, had no significant stimulatory effect upon the recompaction rte of embryos in the reduced calcium medium. In addition, the calcium antagonist Verapamil (0.3 mM), which blocks calcium uptake by cells, significantly inhibited recompaction in standard culture medium. Large doses of diazepam inhibited recompaction only slightly in standard culture medium. Large doses of diazepam inhibited recompaction only slightly in standard culture medium. We conclude that calcium uptake into the cytoplasm is required for recompaction, but that cell surface calcium is also required and is rate-limiting under these experimental conditions.     

A gene function required for cell cycle progression during the G1 portion of the cell cycle and for maintenance of macronuclear DNA synthesis in Paramecium tetraurelia

The ccl mutation in Paramecium tetraurelia reversibly and rapidly blocks cell cycle progression and DNA synthesis at the restrictive temperature. Progression through the cell cycle is blocked during both the G1 and S portions of the cell cycle, while at the restrictive temperature there is neither residual cell cycle progression nor induction of excess delay of subsequent cell cycle events. DNA synthesis activity is reduced to 50% of the normal level in about 5 min and is completely blocked at 30 min after a shift to restrictive temperature. On return to permissive conditions, DNA synthesis is reactivated with similar kinetics.     

Co-dominant markers for the selection of somatic dihybrids and polyhybrids

Resistance to ouabain and puromycin are shown to represent very useful co-dominant characters for the selection of somatic hybrids between mammalian cells, after fusion with polyethylene glycol. We therefore used, with success, a number of Chinese hamster and mouse cell lines carrying these markers in association with thymidine kinase and hypoxanthine-guanine-phosphoribosyl transferase deficiency for selection of hybrids of triparental origin.     

A sodium requirement for mitogen-induced proliferation in human peripheral blood lymphocytes

Mitogen-stimulated DNA synthesis in human peripheral blood lymphocytes is dependent on extracellular Na. DNA synthesis was similarly inhibited in:

1. 1. Cells that were suspended in hypotonic media containing decreased extracellular Na.

2. 2. Cells that were suspended in media containing decreased Na and equimolar replacement with choline.

3. 3. Cells that were suspended in media containing decreased Na and equiosmolar replacement with mannitol.

A decreased PHA-induced DNA synthesis was observed at day 3 even when lymphocytes were exposed to low Na for only the first 3 h and then returned to normal levels of Na. Our studies of protein synthesis indicate that the effect of lowered extracellular Na on DNA synthesis and cell division is not due to an initial inhibition of overall protein synthesis. These data suggest that reduced external Na has a significant effect on some specific early event(s) (3 h) in lymphocyte mitogenesis.     

Evidence for the association of two cell surface glycoproteins of 13762 mammary ascites tumor cells : Concanavalin A-induced redistribution of peanut agglutinin-binding proteins

The behavior of the cell surface concanavalin A (conA) receptors and of peanut agglutinin (PNA) receptors on the MAT-B1 ascites subline of the 13762 rat mammary adenocarcinoma was examined using fluorescein-labeled conA and PNA. ASGP-1, the major glucosamine-containing glycoprotein of these ascites cells, is the only PNA-binding protein observed by dodecyl sulfate electrophoresis. ASGP-2, the second most prominent component after glucosamine labeling, is the most abundant conA-binding protein. These two glycoproteins were previously shown to be associated as a complex in detergent extracts of the cells [20]. ConA-binding proteins, upon incubation with fluorescein-labeled conA (FITC-conA), redistribute on the cell surface into small and large aggregates similar, but not identical, to those seen in ‘patching’ and ‘capping’ experiments with lymphocytes. PNA-binding proteins failed to redistribute during incubation with fluorescein-labeled PNA (FITC-PNA) and appeared in a diffusely stained pattern around the circumference of the cells. However, when cells were treated with unlabeled conA followed by FITC-PNA, or with FITC-PNA followed by unlabeled conA, there was marked redistribution of the FITC-PNA. These results indicate that ASGP-1 redistributes in response to the movement of conAbinding proteins and supports our hypothesis that ASGP-1 and ASGP-2 are associated on the plasma membrane at the cell surface as well as in detergent extracts.     

Mitochondrial DNA synthesis in overmatured eggs of the starfish

DNA synthesis was studied during the overmaturational stage of starfish eggs. Some DNA synthesis took place in the overmature eggs of the starfish. The DNA synthesis was resistant to aphidicolin. Judging from its circularity and small molecular size, the DNA synthesized is of mitochondrial origin.     

Comparative study of the cellular replication kinetics of rat mammary epithelial cells in vivo and in vitro

Ts-131b, one of the temperature-sensitive (ts) mutants isolated from mouse FM3A cells, was found to be defective in DNA replication at a non-permissive temperature. After the cells were transferred to 39.5 °C, the cell number increased by only 10% and the rate of incorporation of precursors into cellular DNA decreased rapidly. Cell cycle analysis by a flow cytometric method with the cells incubated at 39.5 °C revealed that progression of the cells through the S phase was inhibited and most of the cells were arrested in the S phase. To study the defect in DNA replication of this ts-mutant at 39.5 °C, DNA-fiber autoradiography was performed to measure the rate of DNA-chain elongation. The results showed that the rate of DNA-chain elongation was decreased at 6 h after the temperature shift. However, since the decrease in the rate of DNA-chain elongation was not sufficient to account for the decrease in the rate of incorporation of the precursors, it was suggested that there was also a decrease in the rate of initiation of DNA replication at some of the replicon origins.     

Increased uptake of thymidine in the activation of sea urchin eggs. II. Cooperativity with phosphorylation, involvement of the cortex, and partial localization of the kinases

Uptake and phosphorylation of exogenously supplied thymidine are stimulated in Strongylocentrotus purpuratus eggs after fertilization. Before fertilization, the rate of uptake is low and less than 10% of the thymidine entering the egg is phosphorylated. After fertilization, the rate of uptake increases over 50-fold and greater than 90% of the thymidine is immediately phosphorylated. These results imply that there is close cooperativity between fertilization-induced uptake and phosphorylation of thymidine. To gain insight into the structural basis of this apparent cooperativity and to provide a partial localization of the kinases, uptake and phosphorylation were measured in centrifuged eggs, and in centrifuged nucleate and anucleate merogons. Electron micrographs show that in these cells, the inner cytoplasmic contents are stratified according to density and displaced within the egg, whereas the outer cortical region of the cytoplasm remains intact. Uptake and phosphorylation of thymidine are fully stimulated in these eggs and merogons after fertilization, suggesting that both processes are mediated by an intact egg cortex. In support of this suggestion, we report that controlled disruption of the egg cortex prior to fertilization by treatment with cytochalasin B (CB) significantly reduces the rates of uptake and phosphorylation after fertilization. The full stimulation of phosphorylation in nucleate and anucleate merogons eliminates any localization of the catalyzing enzymes (thymidine kinase and thymidylate kinase) in the maternal nucleus and other inner cytoplasmic contents differentially segregated by centrifugation.     

Resolution of mitochondrial DNA structures in the large yeast Wickerhamia fluorescens

DAPI (4′6 Diamidino-2-phenylindole) staining of the large yeast Wickerhamia fluorescens revealed extensive cytoplasmic staining material attributable to mitochondrial DNA (mtDNA), often present in large network-like structures. The maintenance of this mtDNA appears to be insensitive to a variety of mitochondrial specific mutagens, suggesting that W. fluorescens may be classified as a petite negative yeast. Restriction enzyme analysis generated a unit genome size of 42(10⁶) D for this mtDNA which, together with determinations of the average mtDNA per cell, allowed an estimate of the cellular copy number of mitochondrial genomes. A physical map of this mtDNA was also derived. These experiments suggested models which might reflect the cytological structures resolved by DAPI staining in W. fluorescens relative to other yeasts.     

Studies on the hexosaminidases from concanavalin A-resistant and sensitive cell lines : A model system for the study of secretion and uptake of lysosomal enzymes

Parental wild-type and concanavalin A (ConA)-resistant Chinese hamster ovary cells (C^R-7) grown in suspension and on the surface of glass culture bottles were analysed for intracellular and extracellular hexosaminidase forms. When intracellular enzymes were examined three forms were identified (designated Hex I, II and III) depending on the conditions used to elute the enzymes from DEAE-cellulose columns. Only Hex I and III were detected in the medium of the cultured cells suggesting that Hex II is not secreted. Several differences in intracellular and extracellular levels of hexosaminidases were found between C^R-7 and parental wild-type cells which are explained by postulating that there is a difference in the relative abilities of these cells to internalize the hexosaminidase forms. This view is supported by results obtained from previous biochemical experiments carried out with these cell lines.     

Receptors for hyaluronate on the surface of parent and virus-transformed cell lines : Binding and aggregation studies

Two sets of parent and virus-transformed cell lines (3T3 vs SV-3T3; BHK vs PY-BHK) were compared with respect to the extent of divalentcation independent aggregation which previously has been shown to depend upon the interaction of endogenous hyaluronate with specific receptors on the cell surface. When measured under conditions of physiological ionic strength, a significant amount of hyaluronidase-inhibitable aggregation was found in the virus-transformed cell lines (SV-3T3 and PY-BHK) but not in their parent counterparts (3T3 and BHK). However, when the same experiment was performed in a high ionic strength solution (0.5 M NaCl), the hyaluronidase inhibitable aggregation was detected in all of the cell lines. The differences in the aggregation between the various cell lines was also reflected in the binding of [³H]hyaluronate. In physiological saline, the virus-transformed cells bound greater amounts of hyaluronate (higher B_max) with a greater affinity (lower k_d) than did their untransformed counterparts. Increasing the ionic strength to 0.5 M NaCl increased the binding of [³H]hyaluronate by each cell line; however, the relative differences between the cell lines remained. These results indicate that variations in the ability of the cells to bind hyaluronate can partially account for the differences between the parent and the virus-transformed cells with respect to their ability to aggregate.     

Immunolocalization of the 33 kD protein in the microvilli of rabbit small-intestinal epithelial cells

Rabbit small-intestinal microvilli isolated by a Ca²⁺ precipitation method contain a 33 kD protein, which has not been observed in microvilli isolated in the presence of Ca²⁺-chelators. The intracellular localization of this protein in rabbit intestinal epithelial cells was studied by immunofluorescence and immunoperoxidase microscopy, and was compared with that of aminopeptidase M, a well-known microvillus membrane-bound enzyme. The results obtained show that the 33 kD protein is located in the inside of the microvillus, but not in the terminal web of the epithelial cell. The protein may also be located on the basolateral surface of the cell.     

The culture of chick embryo chondrocytes and the control of their differentiated functions in vitro. I. Characterization of the chondrocyte-specific phenotypes

We have maintained chick embryo chondrocytes in culture for more than 2 months, passaging the floating cells in the absence of ascorbic acid. Throughout the culture period some of the cells attached to the dish, assuming an epithelial-like morphology and subsequently giving rise to new floating cells. The interconversion of the two cell populations was highest in primaries and decreased with the aging of the culture. Cartilage cells synthesized pro-alpha 1 (II) collagen and sulphated proteoglycans in vitro; compared with floaters, the epithelial-like cells secreted relatively large amounts of fibronectin. When ascorbic acid was added to the medium, all cells attached, maintaining their rounded shape; in this condition the pro-alpha, (II) collagen was matured and collagen fibres were detectable outside the cells. Other specific proteins synthesized by the chondrocytes in culture were also identified. One of these, a 64 K collagenase-sensitive protein, was not related to the type II collagen and may represent a new collagen type.     

Endothelial cells are a site of uptake and degradation of hyaluronic acid in the liver

In a recent communication it was shown that intravenously injected radioactively labelled hyaluronic acid was preferentially taken up by the liver and degraded. We now report that uptake occurs in the liver endothelial cells and that these cells degrade the polysaccharide in vitro into low-molecular weight (LMW) products.     

Modification of the surface ATPase activity in cultured hepatoma cells by lipid-depleted media

Several compounds have been described which elute fibronectin from a gelatin-Sepharose affinity support. In the present study, it has been found that the potent chaotrophic agent, lithium di-iodosalicylic acid, is 20-fold more effective in eluting fibronectin from collagen than any other presently described fibronectin elution agent. Lithium di-iodosalicylic acid and certain other fibronectin elution agents have been characterized in regard to several parameters involved in the elution of fibronectin from collagen and plastic substrata. By assaying for retention of the cell adhesive activity of fibronectin, it has been demonstrated that 8 M urea + 0.1 M citric acid, pH 4.7, is the most effective condition for preservation of biological activity following elution of fibronectin from the gelatin-Sepharose affinity support.     

Tetrahymena calmodulin. Characterization of an anti-tetrahymena calmodulin and the immunofluorescent localization in Tetrahymena

A rabbit antiserum specific for Tetrahymena calmodulin was prepared and characterized: In Ouchterlony's immunodiffusion test, the antiserum gave rise to a single precipitin line only with calmodulin in the reaction with crude Tetrahymena extract and the antiserum cross-reacted with a calmodulin fraction from Paramecium, but not with several calmodulin fractions, from higher organisms. Calmodulins from the ciliates appear to share some antigenic determinants which are absent in calmodulins from higher organisms. The intracellular localization of calmodulin was investigated by indirect immunofluorescent method using anti-Tetrahymena calmodulin antibody purified on an antigen-Sepharose affinity column. Immunofluorescence was localized in the oral apparatus, cilia, basal bodies, the anterior end of the cell, and the contractile vacuole pores. The localization suggested involvement of calmodulin in food vacuole formation (nutrient uptake), excretion of contractile vacuole contents (regulation of osmotic pressure), and in ciliary movement (reversal). The suggestion was supported by the observation that trifluoperazine markedly suppressed food vacuole formation and excretion of contractile vacuole contents and affected the ciliary motion.     

Extent of histone modifications and H1 content during cell cycle progression in the presence of butyrate

The effects of butyrate upon the extents of phosphorylation of histones H1 and H1(0) during cell-cycle progression have been investigated. Chinese hamster (line CHO) cells were synchronized in early S phase and released into medium containing 0 or 15 mM butyrate to resume cell-cycle traverse into G1 of the next cell cycle. Cells were also mechanically selected from monolayer cultures grown in the presence of colcemid and 0 or 15 mM butyrate to obtain greater than 98% pure populations of metaphase cells. Although cell cycle progression is altered by butyrate, electrophoretic patterns of histones H1, H1(0), H3, and H4 indicate that butyrate has little, if any, effect on the extents of H1 and H1(0) phosphorylation during the cell cycle or the mitotic-specific phosphorylation of histone H3. Butyrate does, however, inhibit removal of extraordinary levels of histone H4 acetylation (hyperacetylation) during metaphase, and it appears to cause an increase in the content of H1(0) in chromatin during the S or G2 phases of the cell cycle.