Glutamine synthetase induction in embryonic neural retina. Interactions of receptor-hydrocortisone complexes with cell nuclei.

In the neural retina of the chick embryo, hydrocortisone (HC) elicits differential gene expression resulting in the induction of glutamine synthetase (GS), which is an enzyme marker of differentiation in the retina. The relationship between nuclear binding of receptor-hydrocortisone (R-HC) complexes and GS induction was investigated in cultures of retina tissue from 12-day chick embryos. The number of HC binding sites in the cytoplasm was estimated as 1650+/-200 per retina cell; there are approximately 1500+/-100 acceptor sites for R-HC per retina nucleus. GS induction in the retina became detectable only after R-HC bound to more than 40% of the nuclear acceptors sites; increased binding coincided with higher induction levels, until complete site saturation was attained; Proflavine, which blocks preferentially and completely GS induction in the retina by interfering in the nucleus with the enzyme-inducing action of the hormone, reduced nuclear binding of R-HC by only 20%; thus, only part of the R-HC that binds in the nucleus appears to be directly involved in eliciting the induction of GS. Within one hour after exposure of the retina to an inducing dose of HC, there was translocation of HC and HC-receptors (as R-HC complexes) from the cytoplasm into the nucleus and saturation of nuclear accepegan to decline; in 12 h, it was reduced to 50% of the initial saturation level. Since, during this time, the enzyme activity to increase, persistence of the induced state depends on association of the hormone with only a portion of the sites in the nucleus to which it can bind. The decrease in the amount of bound HC in the nuclei of induced cells was accompanied by an increase in the level of HC receptors in the cytoplasm. About 50% of this increase could be prevented by cycloheximide; this suggests that the reappearance of HC receptors in the cell cytoplasm may be due, at least in part, to de novo synthesis of HC receptors.     

Features of the damage produced by proflavine on transforming deoxyribonucleic acid.

Proflavine formed a complex with transforming deoxyribonucleic acid (DNA) from Haemophilus influenzae, with optimal formation at a ratio of proflavine to DNA of 0.06. The rate of dissociation of the complex by dialysis increased in the order: native, denatured, renatured DNA. The transforming activity of the DNA was reduced by its interaction with proflavine. This inactivation was dependent on the physical state of the DNA, the proflavine concentration, and the temperature. DNA that had been denatured and renatured was most sensitive; native DNA was much less sensitive. The inactivation remained after dialysis and was stable to prolonged storage. It is concluded that the inactivation of transforming DNA by proflavine takes place by a mechanism different from that of DNA-proflavine complex formation.     

The induction of glutamine synthetase in cell aggregates of embryonic neural retina: correlations with differentation and multicellular organization

A rapid increase in the synthesis and accumulation of the enzyme glutamine synthetase (GS) in the neural retina of the chick embryo characterizes the functional differentiation and maturation of this tissue. A precocious increase of GS can be induced in the embryonic retina by hydrocortisone and related corticosteroids. This paper presents evidence that the responsiveness of neural retina cells to GS induction by the hormonal inducer is dependent on histotypic associations and organization. This was demonstrated, using retina from embryos of different ages, by comparing GS induction in cultures of intact retina tissue with that in aggregates of retina cells and in monolayer cultures of retina cells.     

Proflavine Uptake and Release in Sensitive and Resistant Escherichia coli

Both Escherichia coli B and a proflavine-resistant mutant, E. coli B/Pr, took up the same amounts of proflavine when suspended in buffer containing the dye. In growth media, however, sensitive cells took up more proflavine than did resistant cells. Adding growth media or any one of several constituents of these media, including amino acids, glycerol, pyruvic acid, and metabolizable sugars, to resistant cells that had taken up proflavine in buffer caused them to lose the dye, but had less or no effect on sensitive cells. Certian salts caused an equal release of proflavine from resistant and sensitive cells. Proflavine released from resistant cells by glucose was not changed chemically. The effects of temperature and metabolic inhibitors suggest that proflavine uptake is a passive process but that its release may be an active one, dependent on metabolism. Glucose had more effect on the proflavine binding of E. coli B grown in a minimal medium than on that of cells grown in a complex medium. E. coli B was less susceptible to proflavine when growing in a minimal medium. The effects of other synthetic media on proflavine susceptibility of E. coli B were also studied. Deoxyribonucleic acid and envelopes from sensitive and resistant cells bound the same amounts of proflavine, and no difference was seen in the site of dye binding when sensitive and resistant cells that had taken up proflavine in buffer were sonically broken and fractionated. The results suggest that sensitive and resistant cells are equally permeable to proflavine but differ in the ease with which metabolites cause them to release bound proflavine. So far, however, these differences do not account completely for the ability of resistant cells to grow in high proflavine concentrations.     

Cortisol receptors and inducibility of glutamine synthetase in embryonic retina

Glutamine synthetase (GS) is a marker enzyme for Müller glia cells in neural retina. In chick embryo retina GS begins to increase sharply on the 16th day of development, but can be precociously induced by premature supply of the inducer, cortisol, already on the 8th day. At this stage GS inducibility is low, but it increases progressively with embryonic age. We investigated whether there was a corresponding age-dependent increase of cortisol-binding molecules (cortisol receptors) and found that their level is highest in the early retina and decreases with development. In light of this inverse relationship, we examined whether functional characteristics of these receptors change with age, but detected no differences. In in vitro tests, receptors from older retina translocated cortisol into nuclei from young retina, and vice versa, with similar effectiveness. Also, cortisol receptors from liver cells (which differ from retina receptors) can translocate the hormone into retina nuclei, and vice versa. These findings indicate that translocation of cortisol receptors is neither tissue-specific or age-dependent, nor is it conditional on the total amount of receptors normally present in cells. Therefore, the age-dependent increase of GS inducibility in embryonic retina cannot be directly related to quantitative or functional differences of cortisol receptors and is evidently controlled primarily at the gene level. The very large amount of cortisol-binding molecules in early embryonic retina raises the possibility that they play some role in early differentiation of retina cells unrelated to hormone binding.     

The effect of proflavine on HeLa cells

1. The effect of proflavine on the metabolism of RNA, DNA and protein of HeLa cells was studied. 2. The synthesis of RNA, DNA and protein was progressively inhibited by concentrations of proflavine up to 43mum. 3. There was no simple relationship between the degrees of inhibition of synthesis of RNA, DNA and protein by increasing concentrations of proflavine: the synthesis of RNA was most readily inhibited, and the synthesis of protein was relatively insensitive. 4. A concentration of 22mum-proflavine inhibited synthesis of RNA and DNA and caused a progressive loss of RNA from both nucleus and cytoplasm without any accompanying loss of DNA or dry weight from the cells. 5. The rapidly labelled RNA in the nucleus was preferentially degraded and was not transferred in a stable form to the cytoplasm.     

Immunohistochemical Localization of Glycogen Synthase and GSK3β: Control of Glycogen Content in Retina

Glycogen has an important role in energy handling in several brain regions. In the brain, glycogen is localized in astrocytes and its role in several normal and pathological processes has been described, whereas in the retina, glycogen metabolism has been scarcely investigated. The enzyme glycogen phosphorylase has been located in retinal Müller cells; however the cellular location of glycogen synthase (GS) and its regulatory partner, glycogen synthase kinase 3β (GSK3β), has not been investigated. Our aim was to localize these enzymes in the rat retina by immunofluorescence techniques. We found both GS and GSK3β in Müller cells in the synaptic layers, and within the inner segments of photoreceptor cells. The presence of these enzymes in Müller cells suggests that glycogen could be regulated within the retina as in other tissues. Indeed, we showed that glycogen content in the whole retina in vitro was increased by high glucose concentrations, glutamate, and insulin. In contrast, retina glycogen levels were not modified by norepinephrine nor by depolarization with high KCl concentrations. Insulin also induced an increase in glycogen content in cultured Müller cells. The effect of insulin in both, whole retina and cultured Müller cells was blocked by inhibitors of phosphatidyl-inositol 3-kinase, strongly suggesting that glycogen content in retina is modulated by the insulin signaling pathway. The expression of GS and GSK3β in the synaptic layers and photoreceptor cells suggests an important role of GSK3β regulating glycogen synthase in neurons, which opens multiple feasible roles of insulin within the retina.     

Mechanism of enhancement of polynucleotide binding to cells by mutagens.

The binding of polyuridylate to cells is substantially increased by proflavine. This enhanced binding is saturable with respect to time and to the concentration of both proflavine and polyuridylate. Enhancement is observed only when cells are exposed to both proflavine and polyuridylate together and depends cooperatively on the proflavine concentration. The resulting complex formed between the cell, proflavine, and polyuridylate can be dissociated with salt but not with sucrose solutions. An increase in the binding of polyuridylate to cells similar to that observed with proflavine was also obtained with cationic dyes such as acridine orange, 9-aminoacridine, and Hoechst 33258, while the introduction of a bulky polysaccharide residue, dextran, into the dyes cancels these effects. Similarly, cationic aromatic compounds such as primaquine and quinacrine which carry bulky nonplanar substituents or aliphatic cationic compounds like ethylenediamine do not enhance binding. Proflavine is unable to augment the binding of a basic macromolecule, diethylaminoethylaminoethyldextran, to cells. The model proposed for the enhanced binding of polyuridylate is based on the cooperative formation of stacked complexes of cationic dye located between the cell surface and the bound polyuridylate.     

Effects of cytosine arabinoside on differential gene expression in embryonic neural retina. II. Immunochemical studies on the accumulation of glutamine synthetase

Cytosine arabinoside (Ara-C) elicits a significant increase in the level of the enzyme glutamine synthetase (GS) while it markedly reduces overall RNA and protein synthesis in cultures of embryonic chick neural retina. This increase was analyzed by radioimmunochemical procedures and compared with the induction of GS by hydrocortisone (HC). Accumulation of GS in Ara-C-treated retinas was found to be due to de novo synthesis of the enzyme; however, unlike the induction of GS by HC, Ara-C caused no measurable increase in the rate of GS synthesis. The results indicate that Ara-C facilitates GS accumulation largely by preventing degradation of the enzyme. Even though Ara-C inhibits the bulk of RNA synthesis in the retina, it does not stop the formation of GS-specific RNA templates. However, the progressive accumulation of these templates does not result in an increased rate of GS synthesis unless Ara-C is withdrawn from such cultures under suitable experimental conditions. Thus, it is suggested that the continuous presence of Ara-C imposes a reversible hindrance at the translational level which limits the rate of GS synthesis. The results demonstrate that the increase in retinal GS elicited by Ara-C is achieved through mechanisms which are quite different from those involved in the hydrocortisone-mediated induction of this enzyme.     

Hormonal induction of glutamine synthetase in cultures of embryonic retina cells: requirement for neuron-glia contact interactions

Cortisol induces glutamine synthetase (GS) in gliocytes of chick embryo neural retina. Using adherent cultures of retina cells we have demonstrated that responsiveness of the gliocytes to GS induction by the hormone requires contact with neurons. GS is not inducible in high-density cultures depleted of neurons and consisting only of gliocytes. In neuron-containing cultures, induced GS was detected immunohistochemically only in those gliocytes that were closely juxtaposed with clusters of neurons. Unlike the induction of GS, the expression of carbonic anhydrase-C (which does not require cortisol) persisted in these glia cells also in the absence of neurons. The nature and role of glia-neuron interactions in the hormonal induction of GS are briefly discussed.     

Expression and glycogenic effect of glycogen-targeting protein phosphatase 1 regulatory subunit GL in cultured human muscle

Glycogen-targeting PP1 (protein phosphatase 1) subunit G(L) (coded for by the PPP1R3B gene) is expressed in human, but not rodent, skeletal muscle. Its effects on muscle glycogen metabolism are unknown. We show that G(L) mRNA levels in primary cultured human myotubes are similar to those in freshly excised muscle, unlike subunits G(M) (gene PPP1R3A) or PTG (protein targeting to glycogen; gene PPP1R3C), which decrease strikingly. In cultured myotubes, expression of the genes coding for G(L), G(M) and PTG is not regulated by glucose or insulin. Overexpression of G(L) activates myotube GS (glycogen synthase), glycogenesis in glucose-replete and -depleted cells and glycogen accumulation. Compared with overexpressed G(M), G(L) has a more potent activating effect on glycogenesis, while marked enhancement of their combined action is only observed in glucose-replete cells. G(L) does not affect GP (glycogen phosphorylase) activity, while co-overexpression with muscle GP impairs G(L) activation of GS in glucose-replete cells. G(L) enhances long-term glycogenesis additively to glucose depletion and insulin, although G(L) does not change the phosphorylation of GSK3 (GS kinase 3) on Ser9 or its upstream regulator kinase Akt/protein kinase B on Ser473, nor its response to insulin. In conclusion, in cultured human myotubes, the G(L) gene is expressed as in muscle tissue and is unresponsive to glucose or insulin, as are G(M) and PTG genes. G(L) activates GS regardless of glucose, does not regulate GP and stimulates glycogenesis in combination with insulin and glucose depletion.     

Reciprocal regulation of adult rat hepatocyte growth and functional activities in culture by dimethyl sulfoxide

Hepatocyte DNA synthesis, initiated by epidermal growth factor (EGF), is reversibly inhibited by 2% dimethyl sulfoxide (DMSO). At that concentration, both the survival of the cells in culture and the expression of differentiated functions are prolonged. DMSO does not affect thymidine uptake or EGF receptor binding. Moreover, EGF receptor binding is maintained at 84% of initial 12 hr binding when cells are cultured for several days in the presence of DMSO, whereas specific receptor binding declines to 49% of initial binding under standard culture conditions without DMSO. Studies of hepatocyte functional activity indicate that, during early culture, total cellular export protein synthesis, specific albumin synthesis, and glycogen synthesis are enhanced in the presence of DMSO. Dexamethasone is required for the effect of DMSO on survival, and although dexamethasone alone enhances hepatocyte DNA synthesis in the presence of EGF, it does not reverse the inhibitory effect of 2% DMSO on DNA replication. The correlation of prolonged survival with growth inhibition supports the hypothesis that hepatic growth and differentiated functional activity may be reciprocally regulated.     

The pattern of DNA methylation in the delta-crystallin genes in transdifferentiating neural retina cultures

Terminally differentiated lens fibre cells are formed in the vertebrate lens throughout life. Lens fibre cells may also be obtained by an in vitro process termed transdifferentiation, from certain tissues of different developmental origin from lens, such as embryo neural retina. delta-Crystallin is the major protein in the chick embryo lens fibre cells, and also in transdifferentiated lens cells obtained from cultured embryonic neural retina. Lens crystallin proteins and mRNA are present at low levels in the intact embryonic neural retina but are no longer detectable in the early stages of neural retina cell culture. However, levels rise steeply in the later stages and crystallins become the major products in terminally transdifferentiating neural retina cultures. We have used this system to test the hypothesis that the patterns of DNA methylation in particular genes are correlated with gene expression. A number of developmentally regulated genes have been found to be undermethylated in tissues where they are expressed, and methylated in tissues where they are not. However this correspondence does not always hold true. Eight-day-old embryonic neural retina was cultured for the period of time during which crystallin gene expression increases 100-fold. DNA methylation in the delta-crystallin gene region was analysed at several stages of cell culture by using the restriction endonucleases HpaII and MspI which cleave at the sequence CCGG. The former enzyme cannot cleave internally methylated cytosine (CmCGG) while the latter cannot cleave externally methylated cytosine (mCCGG). We detect no change in the methylation of CCGG sites within the delta-crystallin gene regions during transdifferentiation. Since dramatic changes in delta-crystallin gene expression occur during this process we conclude that large scale alterations in the pattern of DNA methylation are not a necessary accompaniment to changes in gene activity.     

Reversibility of muscle differentiation in the absence of commitment: analysis of a myogenic cell line temperature-sensitive for commitment

The interrelationship between commitment (irreversible withdrawal from the cell cycle) and muscle-specific gene expression was analyzed with the myogenic cell line ts 3b-2, which is temperature sensitive for commitment and cell fusion. The rates of synthesis and levels of accumulation of muscle-specific mRNAs and proteins in the ts 3b-2 cells at permissive and nonpermissive temperatures are comparable, indicating that neither commitment nor cell fusion is required for induction of muscle-specific gene expression. In the absence of commitment, the cells are reversibly withdrawn from the cell cycle during gene induction, and expression of the muscle-specific genes is deinduced upon the switch to growth-stimulating conditions. The deinduction reflects coordinate and preferential cessation of muscle-specific mRNA synthesis, coupled with destabilization of the muscle-specific mRNAs in the cytoplasm, without effect on constitutively expressed housekeeping protein genes. The phenotype of the ts 3b-2 cells demonstrates that commitment and muscle-specific gene expression are both required, but alone are insufficient, to produce the terminally differentiated muscle phenotype.