Excretion of spermidine from BHK-21/C13 cells exposed to 6-thioguanosine.

The growth of BHK-21/C13 cells in monolayer cultures was inhibited by 6-thioguanosine. Accumulation of putrescine, spermidine, and spermine was inhibited by 6-thioguanosine, and cells incubated in the presence of the drug had a decreased content of polyamines relative to control cells. These effects were more marked for spermidine than for spermine or putrescine. Consequently, the intracellular spermidine:spermine molar ratio was decreased in cells exposed to the drug. Cells, the polyamines of which had been labeled with [3H]putrescine, were incubated in the presence or absence of 6-thioguanosine. More polyamines were lost from cells exposed to the drug than from control cells. The radioactive material excreted was predominantly spermidine, both as its free form and in a conjugated form, even when the cells contained large amounts of labeled spermine. This release of spermidine from BHK-21/C13 cells into the culture medium was a specific response of the cells to alterations in growth potential rather than a consequence of leakage due to cell lysis.

Subcellular location and growth stage dependence of the DNA polymerases of Euglena gracilis.

The subcellular location and growth stage dependence of the DNA polymerases of Euglena gracilis strain Z and of a bleached derivative of the strain have been studied by fractionation of the enzymes from extracts of whole cells and subcellular fractions on DEAE-cellulose. A new method for the rapid isolation of nuclei was employed. Of the major enzymes, pol A has a predominantly nuclear location and pol B a predominantly cytoplasmic location. Pol A is 4-fold and pol B 15-fold more active in exponentially-growing cells than in stationary-phase cells, pol B representing 90% of the combined activities in exponential-phase cells. The activity of the mitochondrial DNA polymerase increases about 3-fold as the cells enter stationary phase while that of the chloroplast DNA polymerase is greater in exponential-phase cells. The chloroplast enzyme persists in cells which have been reversibly bleached. The results are compared to those of similar experiments involving primitive and higher eucaryotes.

Uptake and excretion of polyamines from baby hamster kidney cells (BHK-21/C13). The effect of serum on confluent cell cultures.

In confluent cultures of BHK-21/C13 cells there was little uptake of exogenous polyamines and only a low level of polyamine biosynthesis. These cultures continuously excreted polyamines into the extracellular medium. Spermidine, in both the free and bound form, was the predominant excretion product, whereas the major intracellular polyamine was spermine implying that excretion of polyamines was specific. Reinitiation of growth by the addition of fresh serum immediately increased the uptake of exogenous putrescine, increased the biosynthesis of polyamines and decreased the excretion of polyamines. Thus, polyamine transport into and out of the cell appears to be regulated by the growth status of that cell.

The effect of polyamines of DNA synthesis in vitro.

Nuclei and DNA-dependent polymerases alpha and beta were isolated from exponentially growing baby hamster kidney 21/C13 cells and were used to study the effects of polyamines on DNA synthesis in vitro. The greatest effect was observed with spermine, which inhibited both nuclear DNA synthesis and the activity of partially purified DNA polymerase alpha. At 2.5 mM spermine, the maximum concentration used, we observed 58 and 68% inhibition of DNA synthesis by isolated nuclei and polymerase alpha, respectively. In contrast, spermidine caused a small increase in nuclear DNA synthesis at low concentrations (0.5 mM) and inhibition at higher concentrations (2.5 mM); it had no significant effect on the partially purified polymerase alpha. Neither polyamine had any appreciable effect on polymerase beta activity. The results are consistent with the concept that DNA polymerase alpha catalyses the observed DNA synthesis in isolated nuclei.

Factors affecting polyamine excretion from mammalian cells in culture. Inhibitors of polyamine biosynthesis.

Canavanine, diaminopropane, alpha-methylornithine and methylglyoxal bis(guanylhydrazone) decreased the intracellular polyamine concentrations in growing baby hamster kidney cells. Each of the inhibitors also prevented polyamine efflux into the extracellular medium. Concomitant with the decrease in polyamine excretion was a change in the distribution of polyamines in the extracellular medium. In each case there was a decrease in the amount of radioactivity present as free spermidine and an increase in that found as acetyl polyamines. The magnitude of this shift correlated with the degree of inhibition of excretion. It may be that acetyl polyamines play a role in the regulation of polyamine excretion.

The deoxyribonucleic acid polymerases of non-vertebrate eukaryotes.

DNA-dependent DNA polymerases have now been purified from a number of invertebrate animals, protists, higher plants and fungi. In this article we review the properties of these enzymes and compare them with the better-known enzymes of vertebrate animals and prokaryotes. Three facts emerge. Firstly, plants, protists and fungi contain high-molecular-weight DNA polymerases which may be capable of categorization into two groups on the basis of their properties in vitro. Secondly, no enzyme analogous to the vertebrate polymerase-beta has yet been found in such organisms, and thirdly, many of these enzymes possess associated exonuclease activities like those of the bacterial DNA polymerases. On the basis of these findings, some tentative proposals are made about the evolution of DNA polymerases.

Repair of 4-nitroquinoline-1-oxide-induced DNA damage in normal human cells and cells from classical and variant xeroderma pigmentosum.

The effect of 4-nitroquinoline-1-oxide (4NQO) upon 3 fibroblast cell lines derived from normal and xeroderma pigmentosum subjects have been compared. Excision-deficient XP cells (XP2BI), complementation group G, are nearly 200-fold more sensitive than normal cells to the lethal effect of 4NQO while XP variants (XP7TA), are 2-fold more sensitive. This cytotoxicity correlates with the levels of unscheduled DNA synthesis performed by the 3 cell lines. 4NQO causes a dose-related inhibition of DNA replication in all cell lines. However, newly replicated DNA synthesised immediately after treatment of cells with 4NQO is slightly smaller in XP7TA variant cells than in normal cells receiving the same dose of 4NQO, but DNA fragments in excision-deficient XP2BI are 50% smaller. It is likely that replicon elongation and joining together of newly replicated DNA fragments is dependent upon the excision of certain 4NQO-induced lesions, possibly normally repaired by a 'short-patch' repair process defective in XP2BI.

Fundamental aspects of oestrogens: intracellular receptors and gene activation.

The steroid hormones exert their biological responses through a multi-step process involving regulation of gene expression. The hormone passes across the target-cell membrane from the circulation and enters the cytoplasm where it binds to a specific protein designated the receptor. A conformational change in the receptor ensues and the receptor-hormone complex is translocated to the chromatin in the cell nucleus. Interaction of the receptor-hormone complex with specific regions of the DNA and with specific non-histone chromatin proteins leads to initiation of transcription of new molecules of RNA. The primary RNA transcripts are processed to give mature molecules of messenger-RNA which are transported to the cytoplasm where they combine with ribosomes to form polysomes. The messenger-RNA is translated, by the cytoplasmic machinery of protein synthesis, into various new proteins. These proteins then promote the well-established biological responses associated with each particular steroid. Impairment of any of these steps (for example, lack of production of steroid, absence of receptor, a defect in structure of the receptor) results in a disturbance in or loss of the normal biological response.

PIP: The multistep process through which steroid hormones exert their biological responses involves regulation of gene expression. When a hormone passes across a target cell membrane after exiting the circulation, the hormone enters the cytoplasm of the target cell and binds to a specific protein, which is its receptor. Then, a conformation change in the hormone receptor occurs and the complex of receptor and hormone translocates to the chromatin in the cell's nucleus. Initiation of transcription of new molecules of ribonucleic acid (RNA) follows the interaction of the receptor-hormone complex with specific regions of deoxyribonucleic acid (DNA) and with specific nonhistone chromatin proteins. The primary RNA transcripts are then transported to the cell's cytoplasm. In the cytoplasm the messenger RNA combines with ribosomes and forms polysomes. Then, the messenger RNA is translated, via the cytoplasmic machinery of protein synthesis, and the result is the manufacture of various new proteins. These proteins promote the biological responses associated with each particular steroid hormone. When any of these steps are impaired, be it lack of production of steroid, absence of receptor, or a defect in the receptor's structure, there is a disturbance in or loss of the normal biological response. The role of such impairments in diseases associated with estrogen is discussed also.

DNA polymerases of Euglena gracilis: heterogeneity of molecular weight and subunit structure.

Sedimentation analysis of glycerol-density gradients has shown that freshly purified DNA polymerases A and B (pol A and pol B) of Euglena gracilis have molecular weights of 185,000 (8.7S) and 240,000 (10.3S) respectively. They can aggregate in fresh preparations to give forms of higher molecular weight as shown by gel filtration through Sepharose 6B, but on ageing pol B progressively generates species with sedimentation coefficients of 7.4-7.7S, 6.3-6.5S, 4.8S and finally 3.0S. Pol A apparently behaves in a similar fashion though it is unstable. Exposure of pol A and pol B to high ionic strengths can also cause their breakdown to species with lower sedimentation coefficients. The mitochondrial DNA polymerase is distinct, having a molecular weight of 170,000. It is proposed that pol A and pol B are oligomers of the 3.0S subunit and possibly other dissimilar subunits, with pol B having additional factors conferring upon it its extra catalytic functions.

Diminished excretion of polyamines from BHK-21/C13 cells exposed to methylglyoxal bis(guanylhydrazone).

Methylglyoxal bis(guanylhydrazone) (1,1'-[methylethanediylidine)dinitrilo]diguanidine) inhibited the growth of BHK-21/C13 cells in monolayer cultures. Accumulation of spermidine and spermine was inhibited, whereas the accumulation of putrescine was increased. The intracellular spermidine/spermine molar ratio decreased conly slightly after exposure of the cells to 20 micrometer-methylglyoxal bis(guanylhydrazone) for 1 day. Cells incubated in the presence of the drug released less polyamine into the culture medium that did control cells, the polyamine released consisting almost exclusively of spermidine, both free and as a conjugated form.

Onset of ribosome degradation during cessation of growth in BHK-21/C13 cells.

When BHK-21/C13 cells growing exponentially in 10% serum are transferred to a medium containing only 0.25% serum, cell growth is decreased. After initial changes in RNA synthesis and degradation, protein content of the cultures reaches a plateau and eventually DNA synthesis is arrested. rRNA is relatively stable in exponentially growing cells. Immediately after 'step-down' rRNA degradation commences, but poly(A)-containing RNA does not appear to be degraded any faster than in control cells. Reutilization of RNA precursors has been independently measured and amounts to less than 1%/h for rRNA, insufficient to influence the conclusion that rRNA degradation begins almost immediately after 'step-down'. The degree of reutilization of uridine is much greater for poly(A)-containing RNA than for poly(A)-free RNA.

Excretion of polyamines from baby hamster kidney cells (BHK-21/C13: effect of infection with Herpes Simplex Virus Type 1.

Confluent monolayers of BHK-21/C13 cells excreted increasing amounts of polyamines into the extracellular medium with time. Excretion was specificity of spermidine and was not the result of cell death or lysis. Herpes simplex virus type 1 (HSV-1) completely prevented excretion of polyamines from 2 h after infection of the cells. The virus specificity inhibited the release of free spermidine but not of conjugated polyamines.

The activity of deoxyribonucleic acid polymerase in some species of algae.

1. The activities of DNA polymerase preparations from the algae Euglena gracilis, Chlamydomonas reinhardtii, Chlorella pyrenoidosa, Anabaena variabilis and Anacystis nidulans were measured. The blue-green algae Anabaena and Anacystis contain a 5-20-fold higher activity of the enzyme than do the green algae. DNA polymerases from the blue-green algae show a pH optimum of 9 and prefer a relatively low Mg(2+) concentration (1-3mm). DNA polymerases from the green algae, however, display a pH optimum between 7.5 and 8.5 and an optimum Mg(2+) concentration of 8mm. With all algae, a higher polymerase activity was obtained with denatured salmon sperm DNA as template than with native DNA. All four deoxyribonucleoside 5'-triphosphates must be present for full activity of the polymerases. 2. With one exception, the deoxyribonuclease activities in the preparations, measured under conditions of the DNA polymerase assay, are low compared with corresponding preparations from Escherichia coli. Chlamydomonas extracts contain a high deoxyribonuclease activity. 3. After purification on columns of DEAE-cellulose, the polymerase activity was linear over a wide range of protein concentrations, except for Chlamydomonas preparations, where the observed deviation from linearity was probably attributable to the high nuclease activity. 4. DNA polymerases from all these algae bind strongly to DNA-cellulose; 6-40-fold purifications of the enzyme were obtained by chromatography on columns of DNA-cellulose. 5. The partially purified polymerases of Euglena and Anacystis are heat-labile but become much more heat-stable when tested in the presence of DNA.