Antiproliferative effect of interferon on a Burkitt''s lymphoma cell line

The effect of interferon (IF) on the growth of a Burkitt's lymphoma cell line was analysed. The degree of depression of cell doublings was the same if the cells were in a steady state mode of exponential growth or in a resting state (G0) when IF was added. As IF had a lag time of 24 h before decreased growth could be observed, cells in G0 did not seem to be more sensitive when growth was estimated by cell counts expressed as cell doublings. IF inhibited cells to proceed into the cell cycle and the possibility that IF may increase the escape into a G0 loop is discussed.     

Comparison of glycerophosphate acyltransferases from Euglena chloroplasts and microsomes

The acylation of sn-glycerol 3-phosphate is a common reaction in the pathways leading to the biosynthesis of glycerol-derived phospholipids, galactolipids, and sulfolipids. Enzymes catalyzing this reaction have been solubilized from Euglena chloroplasts, microsomes, and mitochondria (B. A. Boehler and M. L. Ernst-Fonberg (1976) Arch. Biochem. Biophys. 175, 229-235; L. V. Grobovsky, S. Hershenson, and M. L. Ernst-Fonberg (1979) FEBS Lett. 102, 261-264). Some characteristics of the reactions catalyzed by the acyl-CoA:sn-glycerol-3-phosphate O-acyltransferases (EC 2.3.1.15) solubilized from chloroplasts and microsomes of Euglena have been compared. Although the two enzymes have some common features, including stimulation by bovine serum albumin and phosphatidyl choline and sensitivity to sulfhydryl-binding reagents, they differ in their stabilities and responses to salt and glycerol. They exhibit different acyl-CoA substrate dependency curves. The proportions of monoacyl sn-glycerol-3-phosphate acyltransferase activity differ in the two solubilized enzyme preparations, and different products are produced by each of the glycerophosphate acyltransferases solubilized from chloroplasts and microsomes, respectively. Neither glycerophosphate acyltransferase will use palmitoyl- or myristoyl-acyl carrier protein (ACP) as a substrate whereas both use the corresponding CoA esters. Neither is inhibited by ACP, but the enzyme from microsomes is inhibited by CoA.     

Identification of external membrane proteins of the T lymphoblast cell line CCRF-CEM

The 70 membrane proteins of the T lymphoblast cell line CCRF-CEM were characterized by

1. 1. [³⁵S]methionine internal radiolabeling;

2. 2. [¹²⁵I]iodine labeling by a lactoperoxidase-mediated method;

3. 3. [³H]fucose internal labeling;

4. 4. binding to a lentil lectin adsorbant column;

5. 5. susceptibility to digestion with limited amounts of papain.

Of the three methods of radiolabeling membrane proteins, [³⁵S]methionine best displayed all proteins although some individual proteins were heavily iodinated or fucosylated. Thirty proteins were externally exposed as defined by susceptibility to lactoperoxidase-mediated radio-iodination and to digestion with minute amounts of papain. Thirtyfive proteins were bound to a lentil lectin absorbant column. p44 (HLA-A and -B antigens) were iodinated, fucosylated, susceptible to papain digestion and bound to the lectin column. β₂-Microglobulin was iodinated and bound to the lectin column. The identifications and functions of other membrane proteins were not known. In general, proteins of high molecular weight (100 000 to 250 000 D) were more heavily radio-iodinated and fucosylated than were proteins of lower molecular weights. p95 was the most heavily fucosylated protein, p110, which had been identified only on T lymphoblasts, was fucosylated and was iodinated. p65, which was found only on the T lymphoblast line CCRF-CEM and could represent a lymphocyte subpopulation-specific molecule, was iodinated and fucosylated. p15 and p18 were equally and densely labeled with [³⁵S]methionine but only p18 was fucosylated and it was heavily radio-iodinated. These experiments help to define the external membrane proteins of a T lymphoblast cell line in part for the selection of proteins for isolation in order to raise antisera for immunodiagnostic and functional studies.     

Associated effects of sodium butyrate on histone acetylation and estrogen receptor in the human breast cancer cell line MCF-7

Sodium butyrate at a concentration of 5mM causes significant hyperacetylation of the core histones in the human breast cancer cell line MCF-7. Histone hyperacetylation was achieved in rapidly-growing cells at 40% confluency after 24 hours in 5mM sodium butyrate. More nearly confluent cells did not reach as high a level of histone hyperacetylation. Upon assaying the estrogen receptors, both cytosolic and KCl-extractable nuclear, we found that butyrate treatment had lowered the estrogen receptor levels in both compartments. To our knowledge this is the first report of an effect of sodium butyrate on estrogen receptor levels.     

Response of large oocytes of Xenopus laevis to progesterone in vitro in relation to oocyte size and time after previous HCG-induced ovulation.

The injection of Xenopus laevis females with human chorionic gonadotropin (HCG) leads to ovulation (and maturation) of oocytes whose diameters are 1.2 mm or larger. However, when Xenopus oocytes are removed from their follicular investments by manual dissection and exposed to the steroid, progesterone, in vitro, they exhibit maturation down to about 0.90 mm in diameter with the majority larger than 1.0 mm showing a positive response. Within each female the larger of the oocytes undergo maturation earlier than smaller ones.The response of oocytes also was shown to depend on the length of time since females were last stimulated to ovulate. Similar-sized oocytes from recently ovulated (stimulated) females matured much faster than those of untreated, unstimulated females. Indeed, even the smaller oocytes from stimulated females often matured before the largest oocytes of females without previous HCG injection.The experiments demonstrate that the physiological state of an oocyte cannot be accurately deduced solely from its size nor response to gonadotropins; unresponsiveness presumably being due to inability of follicular elements to respond to the trophic hormones or transfer the stimulus to the oocyte via the appropriate steroid.     

Equilibrium constants under physiological conditions for the reactions of the nonphosphorylated pathway of L-serine biosynthesis

The observed equilibrium constants (K_obs) for the reactions of d-2-phosphoglycerate phosphatase, d-2-Phosphoglycerate^3? + H₂O → d-glycerate^? + HPO₄^2?; d-glycerate dehydrogenase (EC 1.1.1.29), d-Glycerate^? + NAD⁺ → NADH + hydroxypyruvate^? + H⁺; and l-serine:pyruvate aminotransferase (EC 2.6.1.51), Hydroxypyruvate^? + l-H · alanine^± → pyruvate^? + l-H · serine^±; have been determined, directly and indirectly, at 38 °C and under conditions of physiological ionic strength (0.25 m) and physiological ranges of pH and magnesium concentrations. From these observed constants and the acid dissociation and metal-binding constants of the substrates, an ionic equilibrium constant (K) also has been calculated for each reaction. The value of K for the d-2-phosphoglycerate phosphatase reaction is 4.00 × 10³m [ΔG⁰ = ?21.4 kJ/mol (?5.12 kcal/mol)]([H₂0] = 1). Values of K_obs for this reaction at 38 °C, [K⁺] = 0.2 m, I = 0.25 M, and pH 7.0 include 3.39 × 10³m (free [Mg²⁺] = 0), 3.23 × 10³m (free [Mg²⁺] = 10^?3m), and 2.32 × 10³m (free [Mg²⁺] = 10^?2m). The value of K for the d-glycerate dehydrogenase reaction has been determined to be 4.36 ± 0.13 × 10^?13m (38 °C, I = 0.25 M) [ΔG⁰ = 73.6 kJ/mol (17.6 kcal/mol)]. This constant is relatively insensitive to free magnesium concentrations but is affected by changes in temperature [ΔH⁰ = 46.9 kJ/mol (11.2 kcal/mol)]. The value of K for the serine:pyruvate aminotransferase reaction is 5.41 ± 0.11 [ΔG⁰ = ?4.37 kJ/mol (?1.04 kcal/mol)] at 38 °C (I = 0.25 M) and shows a small temperature effect [ΔH⁰ = 16.3 kJ/ mol (3.9 kcal/mol)]. The constant showed no significant effect of ionic strength (0.06–1.0 m) and a response to the hydrogen ion concentration only above pH 8.5. The value of K_obs is 5.50 ± 0.11 at pH 7.0 (38 °C, [K⁺] = 0.2 m, [Mg²⁺] = 0, I = 0.25 M). The results have also allowed the value of K for the d-glycerate kinase reaction (EC 2.7.1.31), d-Glycerate^? + ATP^4? → d-2-phosphoglycerate^3? + ADP^3? + H⁺, to be calculated to be 32.5 m (38 °C, I = 0.25 M). Values for K_obs for this reaction under these conditions and at pH 7.0 include 236 (free [Mg²⁺] = 0) and 50.8 (free [Mg²⁺] = 10^?3m).     

The alveolar macrophage: Its capacity to act as an accessory cell in mitogen-stimulated proliferation of guinea pig lymphocytes

The capacity of the alveolar macrophage to act as an accessory cell in PHA-induced lymphocyte proliferation was investigated and compared with that of the peritoneal and peritoneal exudate macrophages in guinea pigs. When lymph node cells were co-cultured with autologous lung cells recovered by airway lavage, the proliferative response to PHA was greatly enhanced over that of lymph node cells alone. In the presence of peritoneal cells or peritoneal exudate (glycogen-induced) cells, the PHA response was intermediate between that of lymph node cells alone and lymph node cells cultured with lung cells. Experiments using purified macrophages (≥98%) as accessory cells demonstrated that the difference observed between lung and peritoneal accessory cells was due to differences in macrophage function. Furthermore, when lymph node cells were cultured in the upper chamber of a double-chambered Marbrook apparatus, PHA-induced proliferation was enhanced only when lung and not peritoneal macrophages were present in the lower chamber. Additional experiments showed that this difference (1) was not an artifact of the thymidine incorporation assay to measure proliferation; (2) was not affected by changing the macrophage-lymphocyte ratio; and (3) was not simply a trephocytic or growth promoting effect of macrophages which could be replaced by 2-mercaptoethanol.These findings show that macrophages from different sources differ in their abilities to act as accessory cells in PHA-induced lymphocyte proliferation. Alveolar macrophages appear to have an enhanced capacity compared to unstimulated and stimulated peritoneal macrophages in this function. At least part of this difference may be due to a difference in the elaboration of soluble factor (s) by macrophages.     

The mechanism of cell elongation during lens fiber cell differentiation

Lens fiber formation is characterized by extensive cell elongation. Earlier studies have shown that lens cell elongation in vitro can occur in the absence of microtubules and is associated with a proportional increase in cell volume. We have previously suggested that lens fiber cell elongation is directly caused by an increase in cell volume. In this report, lenses from 3- and 6-day-old chicken embryos were three-dimensionally reconstructed from serial sections to provide a measure of cell volume and length during various stages of primary and secondary lens fiber formation. In both cases, cell volume was highly correlated with cell length during lens cell elongation. In addition, during primary lens fiber formation, large intercellular spaces between lens vesicle cells disappeared as these cells began to elongate to form lens fibers. Loss of intercellular spaces would be expected if increasing cell volume were responsible for cell elongation. Finally, results of experiments in which the lens capsule was cut with a fine tungsten needle suggested that the capsule was elastic and normally under tension. These findings were used to formulate a model which accounts for the major events in lens morphogenesis based on (1) the regulation of cell volume, (2) the junctions present between lens cells, and (3) the constraint provided by the elasticity of the lens capsule.     

Factors influencing renal cryopreservation. II. Toxic effects of three cryoprotectants in combination with three vehicle solutions in nonfrozen rabbit cortical slices

The [K+]/[Na+] ratio of rabbit renal cortical slices was used to examine, at 25 degrees C, the effects on viability of three cryoprotectant agents (CPA) (dimethyl sulfoxide (Me2SO), ethylene glycol, and glycerol) in combination with three vehicle solutions (Krebs-Henseleit (K-H), solution A, and RPS-2). Viability assessment by [K+]/[Na+] for all test solutions was made after incubating the slices in modified Cross-Taggart solution (C-T). With K-H and solution A, all concentrations of ethylene glycol and glycerol resulted in lowered ratios, whereas with Me2SO, concentrations greater than 1.4 M are required to reduce [K+]/[Na+]. With RPS-2 no decrease in the ratios was found until concentrations greater than 2.8 M were reached for all three CPAs. Binding of Me2SO to albumin, studied using [14C]Me2SO, was inhibited by RPS-2 when compared to K-H. Introduction and removal of Me2SO at 10 degrees C allowed an improvement in viability, at higher Me2SO concentrations, as compared to 25 degrees C.     

Factors influencing renal cryopreservation. I. Effects of three vehicle solutions and the permeation kinetics of three cryoprotectants assessed with rabbit cortical slices

A renal cortical slice model was used to assess the effects on viability of three vehicle solutions-Krebs-Henseleit (K-H), solution A, and RPS-2--at 25 degrees C. After 120 min incubation no differences in [K+]/[Na+] ratios were found. Tracer techniques were used to study the osmotic effects and permeation kinetics at 25 degrees C of three cryoprotectants (dimethyl sulfoxide (Me2SO), ethylene glycol, and glycerol) and the effect of the vehicle solution (K-H or RPS-2) on Me2SO kinetics. It was found that Me2SO was most permeable and ethylene glycol least, and that ethylene glycol had unusual effects which suggest that it may not act as a simple solute. Differences were found when Me2SO was introduced in K-H and RPS-2 that are believed to be related to the binding properties of Me2SO to cell constituents.     

A method for fractionating rat liver cell nuclei on equilibrium density gradients of CS2SO4.

Sonically disrupted nuclei from proliferating liver cells were fractionated in Cs₂SO₄ equilibrium density gradients. Nuclear constituents were concentrated in three bands designated as light band (LB, 1.21 g/cm³), middle band (MB, 1.26 g/cm³), and heavy band (HB, 1.32 g/cm³). Analysis of protein and nucleic acid distribution in gradients suggests preservation of some macromolecular interactions. Studies comparing distributions of radioactively labeled DNA after 1- or 120-min intervals following tritiated thymidine injection indicate enrichment of nascent DNA in LB and MB. This enrichment is sensitive to time and pressure of sonication. Furthermore, DNA-polymerase activity was demonstrated in the gradient fractions following removal of Cs₂SO₄, with most activity once again in the LB and MB. These results suggest this procedure as an initial step in the isolation of an enzymatically active DNA replication complex.     

Bioautography of cell attachment proteins.

A simple method, termed bioautography, is described which permits the visualization of bands of biologically active collagen-dependent cell attachment protein (c-CAP) after gel electrophoresis. The principle of bioautography depends on the “staining” of electrophoretically separated c-CAP's by live mammalian cells. Bioautography demonstrates (a) two bands of cell attachment protein (c-CAP) in serum; and (b) electrophoretic mobility differences between c-CAP's derived from the sera of various mammalian species.     

DNA replication in asynchronous and synchronous ehrlich ascites cells in different conditions of growth

Ehrlich ascites tumour cells were labelled for DNA fibre autoradiography within the peritoneal cavity of a tumour-bearing mouse. The generation and the evaluation of the autoradiographic patterns is described and discussed. To study possible changes of the autoradiographic patterns during a natural S phase the labelling was performed in the mouse or in culture with asynchronous cells which were afterwards separated into synchronous subpopulations by zonal centrifugation. The subpopulations obtained were characterized by flow cytofluorometry in connection with the thymidine labelling index. We compared the DNA fibre autoradiographic patterns of several synchronous and asynchronous cell populations growing in the mouse or under different conditions in culture: The replicon size distributions of all populations examined were virtually the same. The fork movement rate was found to depend mainly on the metabolic condition of the cells. In culture it was significantly slower than in the mouse although a shortened S phase and therewith an increased DNA synthesis rate occurred. During a natural S phase it increased slightly, at most, while the DNA synthesis rate was considerably enhanced at the end of S. The changes in the rate of total DNA synthesis cannot account for the changes in the rate of chain growth. We conclude that the DNA synthesis rate is regulated almost exclusively by changing the replicon initiation frequency, while the fork movement rate is limited by the actual metabolic condition of the cells.     

A glycoprotein involved in aggregation of D. discoideum is distributed on the cell surface in a nonrandom fashion favoring cell junctions

We studied the distribution on the cell surface of a glycoprotein (gp150) involved in the aggregation process of Dictyostelium discoideum. Using immunohemocyanin labeling of intact aggregates and visualization by scanning electron microscopy (SEM), we found a distribution gradient of gp150 wherein the concentration was enriched at or near sites of cell contact. When the distribution of gp150 on the cell surface was examined with immunoferritin and transmission electron microscopy (TEM), we found that gp150 was closely associated with the plasma membrane.     

Inhibition of cell aggregation by specific carbohydrates.

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

The multicomponent nature of teratoma cell adhesion factor

The multicomponent nature of teratoma cell adhesion factor has been demonstrated. Fractionation of crude ascites fluid on a DEAE cellulose ion exchange column shows that two or more components are involved in teratoma adhesion factor (TAF) activity. Glycoproteins (or proteoglycans) in fractionated ascites fluid were localized in polyacrylamide gels. The possible role of these sugar-containing molecules in teratoma cell adhesion and current hypotheses on the mechanism of carbohydrate involvement in intercellular adhesion are discussed.     

Stimulation of differentiation of several murine embryonal carcinoma cell lines by retinoic acid

Retinoic acid stimulates several murine embryonal carcinoma (EC) cell lines, even those previously considered to be incapable of differentiating, to give rise to cell types distinguishable from the parental phenotype in morphology, production of plasminogen activator and surface protein properties. Retinoic acid promotes these changes over a range of low concentrations (10⁻⁹–10⁻⁵ M) which are generally non-toxic to the cells. The effects are clearly demonstrated when EC cells are aggregated prior to exposure to retinoic acid. It is concluded that the observed phenotypic alterations induced by retinoic acid reflect differentiation of the EC cells since non-EC cell characteristics are maintained by cloned cells several generations after retinoic acid is removed from the cultures. Our studies suggest that although retinoic acid stimulates the conversion of EC cells to differentiated derivatives, it does not influence the direction of differentiation. Furthermore, the effectiveness of retinoic acid in stimulating differentiation of EC cells from lines such as Nulli-SCC1 raises the question of whether true ‘nullipotent’ EC lines really exist.     

Analysis of glial cell differentiation in peripheral nervous tissue: II. Neurons promote S100 synthesis by purified glial precursor cell populations

Isolated sensory neurons in vitro do not contain or synthesize S100, whereas glial cell precursor populations do. These precursor cells, when isolated from other cell types, produce low levels of S100 but never undergo the developmental transition to produce high levels of S100. When glial cell precursors are combined with isolated, live or paraformaldehyde-fixed sensory neurons, the precursor cells do undergo the second transition, and accumulate high levels of S100. Peroxidase-anti-peroxidase immunohistochemical staining for S100 confirms previous conclusions (B. Holton and J. A. Weston, 1982, Develop. Biol.89, 64–71) that only those glial cells which are closely apposed to neurons contain augmented levels of S100. This stimulation appears to be specific to neuronal/glial interactions since live or fixed fibroblasts, when cocultured with glial precursor cells, do not promote accumulation of S100 by the glial cells.