James D. Ebert

Changes in membrane potential during the cell cycle

Abstract The membrane potential of isolated synchronized Chinese hamster lung cells (V79) has been determined as a function of their position in the cell cycle. During G 1 the cells exhibit a low but increasing membrane potential which rises sharply at the onset of the S phase. The elevated membrane potential is maintained throughout S and G 2 and declines again when the cells enter mitosis. Membrane potentials in an unsynchronized culture, which was recorded from both mitotic and interphase cells physically associated in groups and clusters, were similar to the plateau level obtained during S and G 2 in isolated synchronized cells, and exhibited little variation. It is concluded that although the membrane potential of isolated cells fluctuates during the cell cycle, it plays no causal role as a regulator of mitotic activity.

External potassium and baby hamster kidney cells: intracellular ions, ATP, growth, DNA synthesis and membrane potential.

Abstract The relationship between cell multiplication, DNA synthesis and external potassium concentration has been investigated in baby hamster kidney (BHK) cells. Alterations in the rate of 3 H-thymidine incorporation were paralleled by changes in the rate of growth. The optimal external potassium concentration was 64 m M and either an increase or decrease from this level was inhibitory. Changes in 3 H-thymidine incorporation with the various potassium media appeared to be immediate (5 min pulse). Intracellular sodium and potassium concentrations measured at 5 min, 2 hr, and 24 hr at various external potassium concentrations indicated that the changes in 3 H-thymidine incorporation were not related to changing intracellular ion levels. The ATP content of potassium-inhibited cells was not significantly different than control cells. The membrane potential was primarily dependent on the potassium distribution and could be manipulated by changing the ratio of external to internal potassium. The relationship between membrane potential and 3 H-thymidine was explored by varying the external potassium concentration from 1.75 to 121 m M . To distinguish between the effects of external potassium per se and the membrane potential, 3 H-thymidine incorporation was measured at 5 or 93 m M external potassium in normal and potassium-depleted cells. The results raise the possibility that the cell membrane potential may influence DNA synthesis.

Multiple effects of ouabain on BHK cells.

Abstract The effects of ouabain on BHK cells have been investigated. Ouabain (4 × 10 −4 M) inhibited growth, reduced the incorporation of 3 H-thymidine and 14 C-amino acids, promoted a loss of cell potassium and gain of sodium, and reduced the membrane potential. Ouabain (1 × 10 −4 M) had very little effect while 2 × 10 −4 M ouabain had an intermediate effect. ATP content was not reduced after 24 h ouabain treatment except at a dose of 10 −3 M. The recovery from ouabain treatment was dependent on the concentration of ouabain and the period of exposure.

Synergism of bacterial lipopolysaccharides and concanavalin A in the activation of thymic lymphocytes

Bacterial lipopolysaccharides (LPS), a category of antigens now widely considered as B cell specific mitogens, enhanced the activation of murine thymic lymphocytes by Concanavalin A (Con A). Thymocytes cultured with LPS and Con A simultaneously display a two- to tenfold enhancement of DNA synthesis throughout a 6-day culture period: LPS alone is not mitogenic. Thymus cells pretreated with LPS for 1 hr respond to ordinarily submitogenic doses of Con A. The responding thymus cell population is ϑ positive an cortisone resistant. The enhancing effect of LPS is obtained only when LPS is introduced before or simultaneously with Con A; the addition of LPS even 20 min after cells were exposed to Con A produces no augmenting effect. LPS appears to “stabilize” the responsiveness of thymocytes to Con A, in that cells incubated in serum-free medium rapidly lose the ability to respond a Con A, whereas LPS treated cells do not suffer a loss in responsiveness.

DNA synthesis by an insoluble chromatin fraction associated with the nuclear membrane of animal cells

Summary An insoluble chromatin fraction prepared from Chinese hamster, HeLa, and baby hamster kidney (BHK) cells is capable of DNA synthesis in the absence of added primer. The DNA in the insoluble chromatin hybridizes with the RNA of Rous sarcoma virus to a substantially greater degree than does the DNA of the soluble chromatin fraction. Studies of the DNA produced in vitro suggest that the insoluble fraction may be a DNA-synthesizing rather than a terminal-repairing system.

Animal Viruses and Embryos

This review illustrates the thesis that the concepts and techniques of animal virology may provide effective measures for analyzing genetic and epigenetic relations in developing cells. The authors discuss first the effects of introducing nucleic acid in the form of virus on the subsequent history of a cell, paying particular attention to the modification of patterns of differentiation and growth by tumor viruses. The question, can viruses be used to study the origin of differences in cell specific properties, is approached by examining the pathologic effects of viruses on embryos. The role of viruses as causative agents in congenital malformations is considered. Viruses are not only agents for modifying the course of development and for detecting when differences arise in cells. Experiments from several disciplines are discussed which focus on the concept of recombination between vertebrate somatic cells. Can intercellular transfer of subcellular units modify patterns of synthesis in embryonic cells? Among the examples treated are the transfer of enzyme forming capacity by epigenetic recombination in bacterial and vertebrate cells; transfer of antibody forming capacity; the selective uptake and exchange of subcellular particles in the chick embryo; and the use of viruses to facilitate transfer of information between vertebrate cells. Finally the application of recent genetic findings, arising from virological and other microbiological studies, to the ontogeny of one class of specific proteins, antibodies, is explored.

A mitochondrial factor that prevents the effects of antimycin A on myogenesis

Abstract Antimycin A, together with a number of other agents which interfere with or inhibit oxidative phosphorylation in isolated mitochondrial systems, also interferes with normal development of myogenic elements in explanted chick embryos and monolayer cultures of muscle. The effects of these agents are quantitative, both morphological and biochemical indices being used, at millimolar to micromolar concentrations. It is concluded that normal growth and maintenance of myogenic elements initially require structural integrity as well as a balanced oxidative state of membrane elements involved in aerobic phosphorylation. A factor isolated from aqueous extracts of chicken liver mitochondria affords protection against antimycin A. Embryos cultivated in a medium containing as much as 0.035 μg of the inhibitor per milliliter for 6 hours, and transferred to normal medium containing the factor, recover. The factor also prevents the action of antimycin A in monolayer cultures of embryonic skeletal muscle. It does not protect embryos against effects of oligomycin. The factor is isolated and purified by passage through a Sephadex G-200 column, followed by adsorption on hydroxyapatite columns and stepwise elution with phosphate buffer. The protein coming off at a concentration of 0.06 M phosphate, pH 7.3, is active and can be treated with light petroleum ether followed by dialysis against 0.002 M phosphate without loss of activity. Activity is lost in 30 minutes at pH 4 (25°C), but at pH 5.5 no reduction in activity is observed. Activity is destroyed by heating at 60°C for 15 minutes. Neither DNase and RNase affect the activity of the factor; it is not destroyed completely by mild trypsin treatment. The factor is antigenic, and, by means of antibodies made to it in rabbits, it has been shown that the factor occurs normally in the early chick embryo.

Levels of Control: A Useful Frame of Perception

Publisher Summary This chapter discusses a common frame of perception or universe of discourse in developmental biology. Development is a series of interlocking processes involving molecular interactions revealed in the communication between nucleus and cytoplasm, in induction, and in the coordination of cell movements. The most fruitful new generalizations of the immediate future are likely to emerge from the frame of perception provided by molecular genetics: the concept of levels of control and their interactions; and the concept of regulation. The importance of agitating existing information, leading to changing concepts, is exemplified by the advances arising out of a reconsideration of the relations between DNA replication and transcription. The chapter tries to carry the generalization a step further, and to examine its implications. The fact that many tissues for example, lens and muscle, undergo rapid cell division prior to “terminal” differentiation raised the question whether new transcription may depend upon an immediately preceding replication. However, it was not until studies of the mechanism of transformation of cells by tumor viruses revealed a requirement for cellular DNA synthesis that the importance of reexamining these relations was brought home.

The differentiation of suppressor cell populations as revealed by studies of the effects of mitogens on the mixed lymphocyte reaction and on the generation of cytotoxic lymphocytes

Abstract The regulation by concanavalin A (Con A) and bacterial lipoloysaccharide (LPS) of the mixed lymphocyte reaction (MLR) and of the generation of cytotoxic lymphocytes (CL) was studied in congenic resistant mice using cortisone resistant thymocytes as the responding cells. LPS enhances the generation of CL selectively when suboptimal numbers of allogeneic cells are present in mixed lymphocyte cultures and also results in the augmentation of the MLR. Mitogenic concentrations of Con A on the other hand suppress the generation of CL regardless of alloantigen dose. The mechanism of suppression cannot be ascribed to the presence of suppressor T cells, since the addition to the cultures of syngeneic cortisone resistant thymocytes activated by Con A does not change the immune response. However, prospective suppressor cells that can be activated by Con A are located in secondary lymphoid organs such as spleen and lymph node. Suppressor activity by those cells is abolished by anti θ plus complement. Con A activated spleen cells suppress the MLR, whereas Con A activated thymocytes amplify the proliferation of responding cells.

Lens culinaris lectin is a T-cell mitogen: Binding inhibition by concanavalin A and phytohemagglutinin-P

Abstract Binding and mitogenicity of a lectin from Lens culinaris (LcH) were studied in mouse lymphocytes. Both continuous and pulse treatment of lymphocytes with LcH induced a mitogenic response selectively in T cells. LcH and Con A, which have similar binding specificities, exhibited binding inhibition both in unfixed cells and glutaraldehype-fixed cells, with native Con A and succinyl Con A and at 37 °C as well as 0 °C. On the other hand, reciprocal binding inhibition by a third T-cell mitogen, phytohemagglutinin-P (PHA-P), was found only in unfixed cells at 37 °C and with native Con A, indicating that the inhibition is a secondary effect as opposed to direct competition for receptors. The inhibition of mitogenic responses to LcH and PHA-P by pretreatment of cells with Con A was studied in relation to the two different types of binding inhibition. Only the type of binding inhibition caused by a secondary effect correlated with interference with the mitogenic response.