In vitro effect of various mitogens on starfish (Asterias rubens) axial organ cells

The effects of various mitogens on axial organ (AO) cells of the sea star have been studied. Pokeweed mitogen (PWM) stimulates [³H]thymidine incorporation by the whole population of axial organ cells of the sea star. This effect occurs 24 hr after the addition of PWM and is maximal at 40 μg/ml. In contrast, no stimulation is observed when coelomocytes are treated with PWM under the same conditions. No stimulation of the whole AO cell population is observed in the presence of Con A or LPS. However, the AO cell population can be divided, on the basis of surface adherence properties, into two subpopulations, adherent and nonadherent. Con A stimulates the nonadherent cells, but not the adherent cells: The stimulating effect is maximal 24 hr after addition of Con A and at 0.2–0.5 μg/ml. In contrast, LPS stimulates the adherent but not the nonadherent cells and the stimulating effect is maximal at 24 hr and at 45 μg/ml.     

Control of cuticle formation by wing imaginal discs in vitro.

Wing discs from late final-instar Ephestia larvae form only pupal cuticle when immediately implanted into pupae which subsequently undergo metamorphosis. However, either pupal or adult structures are made in vitro depending on (1) the ecdysterone dose and/or (2) disc cell proliferation. Continuous culture in ecdysterone (0.5–5.0 μg/ml) results in the appearance of transparent cuticle. On the basis of several criteria, this untanned cuticle is postulated to be scaleless adult cuticle. Discs pulsed with 0.5 μg/ml ecdysterone for 48–120 hr, or with 5.0 μg/ml for 24 hr, formed tanned cuticle. Lower doses of ecdysterone (i.e., 0.5 μg/ml for 24 hr or continuous exposure to 0.05 μg/ml) trigger adult scale formation. Enhancement of [³H]thymidine incorporation by these latter doses suggests the occurrence of disc cell divisions and polyploidization. The choice between pupal and adult pathways by wing discs of this age can be controlled exclusively by ecdysterone; juvenile hormone need not be involved in vitro.     

The formation of the pupal cuticle by Drosophila imaginal discs in vitro

Mass-isolated imaginal discs of Drosophila melanogaster form a chitin-containing pupal procuticle In vitro. Optimal procuticle deposition occurs when the discs are incubated for 4–6 hr with 0.5–1.0 μg/ml of 20-hydroxyecdysone and then with less than 0.05 μg/ml of 20-hydroxyecdysone. The formation of the chitin-containing procuticle is demonstrated using three independent assays: with fluorescene-conjugated cuticle proteins that bind to chitin; by electron microscopy; by incorporation of [³H]glucosamine into a chitin fraction. Synthesis and deposition of pupal cuticle proteins are also demonstrated. Incorporation of [³H]glucosamine into chitin is sensitive to inhibitors of protein, RNA and chitin synthesis, but has little sensitivity to inhibitors of DNA synthesis, and dolichol-dependent glycosylation.     

Modulation of T-cell functions: I. Effect of 2-mercaptoethanol and macrophages on T-cell proliferation

The in vitro effects of 2-mercaptoethanol (2-ME), macrophages (MØ), and concanavalin A (Con A) on the proliferation of normal spleen cells (NSC), MØ-depleted spleen cells (DSC), T cells, T-cell subpopulations, and B cells were assessed by [³H]thymidine incorporation. 2-ME alone was consistently shown not to be mitogenic for purified T cells; however, 2-ME enhanced the early (Days 1 and 2) Con A (2 μg/ml)-induced response of NSC, DSC, and T-cell preparations, but depressed the late response (Days 4 and 5). 2-ME alone was mitogenic for purified B-cells, as reported previously; and the 2-ME-induced B-cell response was inhibited by Con A. Preincubation of T cells with 2-ME was sufficient for enhanced Con A responsiveness; however, if 2-ME was added 24 hr after the initiation of culture, no alteration of the Con A-induced response was observed. Ly-2,3⁺ T cells were unresponsive to Con A (0.3–20 μg/ml), but the addition of 2-ME or peritoneal cells enhanced the Con A responsiveness of Ly-2,3⁺ T cells over 200-fold. Ly-1⁺ T cells responded with a similar doseresponse and kinetic profile as unselected T cells. Although Ly-1⁺ T cells responded to Con A, unlike Ly-2, 3⁺ T cells, extensive removal of MØ significantly reduced the Con A-induced responsiveness of the Ly-1⁺ T cells. The reactivities of Ly-1⁺ and Ly-2,3⁺ DSC could be reconstituted by the addition of MØ or 2-ME; however, the kinetic response of Ly-1⁺ T cells peaked on Day 2–3, and Ly-2,3⁺ T cells had a delayed response which peaked on Day 4–5. The results indicated that (i) 2-ME and/or MØ accelerate the response kinetics of T-cells to Con A; (ii) T-cell subpopulations have differential requirements for MØ and/or 2-ME in the response to Con A; (iii) T-cell subpopulations exhibit differential dose responsiveness to Con A; and (iiii) 2-ME alters Con A responsiveness by a direct effect on T cells.     

Effects of [3H]Udr On the Cell-Cycle Progression of L1210 Cells

Tritium-labelled uridine ([³H]UdR) perturbs progression of L1210 cells through the mitotic cycle. the main effect manifests as a slowdown or arrest of a portion of cells in G₂ and is already observed 2 hr after addition of 0.5–5.0 μCi/ml of [³H]UdR into cultures. At 2.5–5.0 μCi/ml of [³H]UdR a slowdown of cell progression through S is also apparent. Additionally, there is an increase in the number of cells with DNA values higher than 4C in cultures growing in the presence of [³H]UdR for 8–24 hr. A pulse of [³H]UdR of 2 hr duration labels predominantly (95%) cellular RNA. the first cell-cycle effects (G₂ slowdown) are observed when the amount of the incorporated [³H]UdR is such that, on average there are fewer than thirty-six [³H] decays per cell which corresponds to approximately 12–19 rads of radiation. the S-phase slowdown is seen at a dose of incorporated [³H]UdR twice as high as that inducing G₂ effects. the specific localization of [³H]UdR in nucleoli, peripheral nucleoplasm and in cytoplasm, as well as differences in the kinetics of the incorporation in relation to phases of the cell cycle are discussed in the light of the differences between the effects of [³H]UdR and [³H]thymidine. Mathematical modelling of the cell-cycle effects of [³H]UdR is provided.     

Cell movement in somite formation and development in the chick: Inhibition of segmentation

The contribution of active cell movement to somite formation (segmentation) and the later dispersal of the somite sclerotome was examined using cytochalasin D (CD). Stage 14–16 chick embryos were grown over liquid medium. After 8 hr in culture, control embryos had an average of six additional pairs of somites while CD (1–2 μg/ml dissolved in DMSO)-treated embryos had no new somites. DMSO alone had no effect on somitogenesis. CD-treated embryos transferred to drug-free medium recovered and segmentation resumed. Normal and CD-treated segmental plates were examined by SEM. Drug-treated segmental plate cells rounded up, consistent with the interaction of CD on contractile microfilaments. Embryos cultured 8 hr with or without CD were fractured through somite pair 20 and examined by SEM. In untreated embryos the sclerotome had dispersed and was migrating toward the notochord. CD stopped sclerotome dispersal. To test whether CD interfered with elaboration of extracellular matrix material associated with somite development, incorporation of [³H]glucosamine and Na₂³⁵SO₄ by somites and segmental plate was determined. There was no difference in total label incorporation. Molecular-weight profiles of proteoglycan obtained using controlled-pore glass-bead columns showed only small proteoglycans for both treated and control tissues. Therefore, the alteration of segmentation and somite morphogenesis by CD was not due to detectable changes in proteoglycan synthesis.     

Endogenous Regulation of Endothelial Cell Proliferation

Bovine aortic endothelial cells (BAEC) in culture have the ability to regulate their own proliferation. We have found that a fraction below 100,000 daltons obtained from the media of confluent cultures of BAEC inhibits tritiated thymidine [³H]TdR incorporation as well as their proliferation. the inhibition is dose- and time-dependent; maximum inhibition of [³H]TdR incorporation occurs 8 hr after cells are released from synchronization and the inhibitory fraction is added. Inhibition is evident at concentrations as low as 50 μg/ml and reaches a maximum at 600 μg/ml. the blockage of [³H]TdR incorporation is reflected in the inhibition of cell proliferation. In the presence of 400 μg of endogenous inhibitor per ml of media, added at the time of plating, the average population doubling time increases from 19 to 41 hr. These findings indicate that, in culture, BAEC can regulate their own proliferation by synthesizing an endogenous inhibitor(s) of proliferation.     

Chemical N-desulfation of heparin negates its ability to capacitate bovine spermatozoa

Heparin specifically and saturably binds to bovine spermatozoa and stimulates capacitation as assessed by the ability of spermatozoa to undergo a zona pellucida-induced acrosome reaction (AR) in vitro. However, the structural features of heparin important for capacitation are poorly understood The purpose of this study was to determine the importance of the sulfatc content of heparin for its potency to bind to bull spermatozoa and promote agglutination and capacitation. The pyridine salt of heparin was Nndesulfated, which reduced its mean sulfate content from 19.7% to 11.6%. The N-desulfated heparin was then resulfated by incubation with trimcthylamine sulfur trioxide for 6,12, or 24 hr, raising sulfate to original concentrations. Heparin but not N-desulfated beparin competed with [³H]-heparin to bind to spermatozoa. Heparin at 11.6 μg/ml reduced [³H]-heparin binding by half when competing with a saturating concentration of the radidabeled compound (12 μg/ml). N-desulfated heparin did not displace [³H]-heparin. Heparin, resulfated 6 hr or 12 hr, was equal to native heparin in binding potency. Heparin at 50,100, or 250 μg/ml caused more than 40% of the cells to head-to-head agglutinate in aggregates of 8 or more. N-desulfated heparin did not cause agglutination. After spermatozoa were incubated with 0, 5,10, 50, 100, or 250 μg/ml of heparin for 4 hr, 100 μg/ml of lysophosphatidylcholine (LPC)-induccd AR within 20 min in 21.3, 37.7, 27.8, 45.3, 54.2, or 42.5% of the cells, respectively. Sperm exposed to the same concentrations of N-dcsulfated heparin exhibited AR of 17.7,27.3,24.3,22.5,27.7, or 33.8%, respectively, following exposure to LPG Resulfated heparin did not agglutinate or capacitate spermatozoa. In conclusion, N-desulfation of heparin abolished heparin's ability to bind to, agglutinate, and capacitate bovine spermatozoa. Resulfation of N-desulfated heparin restored binding activity but not agglutination or capacitation activity.     

Mechanisms of lymphocyte "deletion" by high concentrations of ligand. I. Cyclic AMP levels and cell death in T-lymphocytes exposed to high concentrations of concanavalin A

DNA synthesis, cell survival, and cyclic AMP (cAMP) levels were compared in whole and purified lymph node cells (LNC) cultured with optimal (5 μg/ml) and excess (200 μg/ml) concentrations of native (N) or succinyl (S) concanavalin A (Con A) as possible models for antigen-induced lymphocyte activation and “high-dose” tolerance. Whole LNC cultured with optimal N-Con A or S-Con A showed continuing DNA synthesis and cell viability between 30 and 50% at 48 hr. In contrast, with excess N-Con A, they showed virtually no [³H]TdR uptake at this time and there was progressive loss of cell viability beginning at 8 hr; by 48 hr almost no viable cells remained. Excess S-Con A induced little cell death up to 24 hr, but by 48 hr only 20% of the cells initially placed in culture remained alive and sythesized DNA. Intracellular cAMP showed a transient rise in cultures stimulated with optimal N-Con A, peaking at 15 min, then returning to normal levels, to rise again between 24 and 48 hr. With excess N-Con A, cAMP rose within 15 min and continued to increase to a peak at 24 hr. cAMP levels in the presence of excess S-Con A remained at control levels for the first 24 hr and increased between 24 and 48 hr. LNC depleted of macrophages and B cells, when cultured in excess N-Con A, had an inhibition of DNA synthesis, elevated cAMP levels, and cell death comparable to whole LNC. It seems unlikely, however, that the increase in cAMP mediates cell killing since cAMP was not elevated yet cell death occurred in nylon wool-purified T cells exposed to excess N-Con A. Dibutyryl cAMP, and prostaglandin E₁, which markedly increase cAMP levels, failed to kill LNC at doses which totally inhibited DNA synthesis, and cells of the mouse T-lymphoma S49 and its cAMP-dependent protein kinase-deficient variant were killed equally by excess N-Con A. It is suggested that a sustained elevation of either cAMP or Ca²⁺ after early commitment may provide a significant mechanism of tolerogenesis.     

EFFECT OF TRITIATED THYMIDINE ON THE KINETICS AND VIABILITY OF 9L CELLS IN VITRO

The colony-forming efficiency of 9L rat gliosarcoma cells was unaffected by treatment with 0.1 μCi/ml of [³H]TdR. However, when cells were treated with 1 or 10 μCi/ml of [³H]Tdr, cell growth was reduced and cell survival decreased. When monolayer 9L cells were treated with 1 μCi/ml of [³H]TdR for up to 72 hr, approximately 5% survived, which is closely related to the percentage of non-cycling cells in this system. When cells were treated with 10 μCi/ml of [³H]TdR for 72 hr, less survival was observed. the additional cell kill observed may be induced by [³H]TdR released from doomed cells into petri dishes during the incubation period of the colony-formation assay.     

Sensitivity of early mouse embryos to [H]thymidine

Effects of intranuclear radiation on the developmental capacity of early mouse embryos were studied by exposing embryos to [³H]thymidine and counting the number of embryos forming blastocysts, trophoblast outgrowths, inner cell masses (ICMs), and two-layer ICMs (differentiated into primary endoderm and ectoderm). When embryos were cultured from the 2-cell stage for 8 days in the continuous presence of [³H]thymidine, concentrations as low as 0.1 nCi/ml reduced the number of embryos forming two-layer ICMs. At 1 nCi/ml, the number of both ICMs and two-layer ICMs was reduced, and at 10 nCi/ml the number of embryos developing to all three post-blastocyst endpoints was reduced. Blastocyst formation was not affected even at the highest concentration tested (100 nCi/ml). When embryos were cultured from the 2-cell stage for 3 days in the presence of [³H]thymidine and then cultured further in unlabelled medium, the effects were similar to those of 8-day exposure. When embryos were exposed to [³H]thymidine for 24 h at various developmental stages, effects were less severe than when they were exposed continuously for 3 or 8 days, and the sensitivity of embryos differed between stages; the lowest concentration that interfered with development was 10 nCi/ml, and exposure at the morula stage was most detrimental to the subsequent development of embryos, particularly that of ICMs. The 24-h exposure of immunosurgically isolated ICMs to [³H]thymidine revealed that the high sensitivity of the ICM to [³H]thymidine persists through the late blastocyst stage and declines progressively thereafter. Autoradiography indicated that the change in radiosensitivity of embryos or ICMs is generally related to their ability to incorporate [³H]thymidine into the DNA.     

Lectin-induced blockage of developmental processes in preimplantation mouse embryos in vitro

This study attempts to assess the developmental importance of cell surface glycoconjugates of preimplantation mouse embryos. This was done by incubating early embryos in various lectins and analyzing subsequent development. If specific cell surface glycoconjugates (lectin receptors) are linked to specific developmental processes, such as cell division, compaction, and blastocyst formation, then different lectins should block these different developmental processes. The results show that wheat-germ agglutinin (WGA; N-acetyl-D-glucosamine-specific) at 50 μg/ml prevents the cell division of four-cell embryos. However, this effect of WGA occurs only in embryos with intact zonae pellucidae. Concanavalin A (Con A; α-D-glucose and α-D-mannose-specific) treatment, 20 μg/ml, of four-cell or early eight-cell embryos prevents compaction, the first major change in cell shape in early mouse embryogenesis. Divalent succinly Con A does not affect development, suggesting that the Con A effect is due to crosslinking of cell surface glycoconjugates. Exposure of four-cell or early eight-cell embryos to 10 μg/ml Lotus Tetragonolobus puprureas agglutinin (LTA; α-L-fucose-specific) or 25 μg/ml Limulus polyphemus agglutinin (LPA; sialic acid-specific) allows compaction or development to the morula stage, but blocks blastocyst formation. All lectins tested retard cell division to some extent. Late morulae and early blastocysts are more resistant than earlier stages to all of the lectins studied. This study demonstrates that very low concentrations of these lectins affect different developmental processes, presumably based upon their sugar specificities.     

Concanavalin A induces neural tissue and cartilage in amphibian early gastrula ectoderm

We have studied in vitro differentiation of explants of the amphibian (Rana temporaria) early gastrula ectoderm after treatment with various concentrations (50–300 μg/ml) of ‘free’ and Sepharose-bound concanavalin A (Con A). The explants were incubated with Con A for 3 h at 20°C; the rolling up of the explants was prevented by using special weights. We have demonstrated that: (1) free Con A has an inducing action on the explants in the concentration range 100–300 μg/ml medium; (2) when treated with Con A the explants produce neural tissue (50–70%), cartilage (20–40%) and, rarely, lentoids (5–10%); (3) the frequency of neural and cartilage inductions was similar at various Con A concentrations; (4) α-methyl-d-mannoside pyranoside inhibited the Con A effects; (5) Sepharose-bound Con A had no effect on the explants, although it was bound to the cell surface of the ectoderm inner layer. Possible mechanisms of the neuralizing and chondrogenic effects of Con A on ectodermal explants are discussed.     

Interferon inhibition of thymidine incorporation into DNA through effects on thymidine transport and uptake

Replenishment of medium after 72 hr of growth of HeLa-S3 cells in dense suspension cultures increased [³H]-thymidine uptake into cells and incorporation into DNA, with the levels reaching a peak ～ 12 hr following medium change; β interferon inhibits the enhanced uptake of [³H]-thymidine and labeling of DNA in a dose-dependent manner. Some reduction in these processes is observed at a concentration as low as 1 u/ml, and ～ 75% inhibition at 640 u/ml. Kinetic analysis has revealed that the rate of labeling of the acid-soluble pool with [³H]-thymidine, measured either at 22°C, or 37°C, is reduced in interferon-treated (640 u/ml, 24 hr) HeLa-S3 cells. At 22°C, the initial rate of thymidine transport at a high (500 μM) thymidine concentration, determined within the first 30 sec of [³H]-thymidine addition was depressed by 44% in interferon-treated HeLa cells. At 37°C, labeled precursors accumulate in acid-soluble material for ～ 8 min after the addition of [³H]-thymidine, after which an apparent equilibrium level is attained. At this temperature, the rate of thymidine uptake and the apparent equilibrium level attained were depressed by 70% in interferon-treated HeLa cells. The reduced incorporation of [³H]-thymidine into DNA in interferon-treated HeLa-S3 cells can be largely explained by interferon inhibition of thymidine transport and phosphorylation.     

Comparison of overall rates of RNA synthesis in implanting and delayed implanting mouse blastocysts in vitro

Implanting and delayed implanting mouse embryos were incubatedin vitro with [³H]uridine for 2–24 hr. The size and specific activity of the [³H]UTP pools were determined by means of a double isotope technique using copolymer synthesis with the [³H]UTP in the embryos, exogenous [¹⁴C]ATP, andE. coli RNA polymerase. Using the rate of incorporation of [³H]uridine into acid-insoluble material and the specific activity of the [³H]UTP pools, it was possible to calculate the overall rate of incorporation of uridine into RNA by the embryos. In implanting embryos it was constant for 24 hr. In contrast, the initial rate of uridine incorporation by the delayed implanting embryos was only 31% of that in implanting embryos (i.e., per cell); this increased steadily during the incubation period, reaching 81% of the rate in implanting embryos after 24 hr. This activation of RNA synthesis by delayed implanting embryosin vitro occurred in the absence of any uterine stimulatory factors. Further, it was shown that although 10% mouse serum would support trophoblastic outgrowthin vitro, it did not influence uptake, distribution of label into nucleotides, or rate of uridine incorporation into RNA in either implanting or delayed implanting embryos. Therefore, it is suggested that if depression and activation of metabolic activity in blastocysts are part of the mechanims of delayed implantation, and if trophoblast outgrowthin vitro is analogous to the process of implantationin vivo, then these two aspects of embryo activation are under different controls.     

Enhancement of concanavalin A-induced vacuolation in macrophages by local anaesthetics (chlorpromazine)

The extensive vacuolation induced in mouse peritoneal macrophages in response to interaction with concanavalin A is markedly enhanced in the presence of chlorpromazine (10^-5 M). At a low concentration of concanavalin A (5 μg/ml) chlorpromazine induces more than double the total number of vacuoles (> 2 μm). At higher concentration of concanavalin A (10–40 μg/ml) though the total number of concanavalin A induced vacuoles is not affected, the size distribution of the vacuoles is changed by chlorpromazine; the number of huge vacuoles (> 5 μm) is doubled. Neither [³H]concanavalin A binding nor its interiorization are affected by the simultaneous presence of chlorpromazine with concanavalin A in the incubation medium. A two-fold increase in chlorpromazine concentration (2·10^-5 M) results in macrophage contraction and inhibition of concanavalin A-induced vacuolation. The data suggest that chlorpromazine affects vacuole formation at the stage of intra-cellular fusion of concanavalin A-bearing pinosomes.     

The effect of tunicamycin on development of the mammalian embryonic pancreas

The role of cell-surface glycoproteins in histogenesis of the embryonic rat pancreas was investigated by studying the effect of tunicamycin (TM) on in vitro development. TM has been shown to block glycosylation of asparagine residues in glycoproteins by inhibiting formation of dolichol oligosaccharide intermediates. Exposure of Day 15 pancreatic rudiments to 1.0 μg TM/ml for 15 or 24 hr inhibited [³H]mannose, [³H]glucosamine, and [³H]fucose incorporation by 95, 85, and 90%, respectively, while [³H]leucine incorporation was reduced by 35%. Similar results were obtained with Day 17 rudiments. These trends were confirmed using sodium dodecyl sulfate-polyacrylamide slab gel electrophoresis and fluorography. Inhibition of [³H]monosaccharide incorporation correlated with reduced binding of RCA I-ferritin conjugates to the cell surface and both effects of TM were reversed by reculturing rudiments in medium lacking the antibiotic. Morphologically, TM treatment resulted in a delay in pancreatic histogenesis and this delay correlated with an inhibition of the normal increase in specific activity of amylase, an acinar cell secretory protein. These effects were not mimicked by treatment with cycloheximide at a concentration which inhibited [³H]leucine incorporation to the same degree observed with TM. The percentage of delayed rudiments decreased as reculturing in the absence of TM was extended.