Susumu Ikegami

Aphidicolin: A specific inhibitor of DNA polymerases in the cytosol of rat liver

Abstract Aphidicolin, a tetracyclic diterpenoid, is known to be antiviral and to inhibit the incorporation of thymidine into DNA of cultured human embryonic lung cells. We examined effects of the compound on the activity of several DNA polymerases obtained from subcellular fractions of rat liver. Aphidicolin at a concentration of 15 μg/ml caused a 85% reduction in level of the activity of crude and partially purified DNA polymerases from the cytosol. However, aphidicolin even at a concentration of 75 μg/ml failed to affect the activity of crude and partially purified DNA polymerases from nuclear and mitochondrial fractions.

Aphidicolin does inhibit repair replication in HeLa cells.

Abstract Aphidicolin was shown to be a specific inhibitor of eukaryotic DNA polymerase α. We have examined the effect of aphidicolin on repair synthesis as well as replication of HeLa cell DNA, and found that it inhibits not only DNA replication but also UV-induced DNA repair in hydroxyurea-arabinosyl cytosine treated cells.

PURIFICATION OF GONAD‐STIMULATING SUBSTANCE OBTAINED FROM RADIAL NERVES OF THE STARFISH, ASTERIAS AMURENSIS

Purification of starfish gonad‐stimulating substance (GSS), which induces shedding of gametes and oocyte maturation, was carried out using lyophilized radial nerves of Asterias amurensis as source material. In the first series of experiments, 1.3 mg of the purified GSS, which induced spawning at a concentration of 0.0096 μg/ml, was isolated from acetone powder of lyophilized radial nerves of 7,360 starfish through several steps of purification procedures consisting of gel‐filtrations on Sephadex G–50 and G–25 columns of various sizes and ion‐exchange chromatography on DEAE‐Sephadex columns by gradient as well as step‐wise elution. With this sample, the molecular weight and amino acid composition of GSS were estimated. Another series of experiments, conducted on a similar amount of material with purification procedures which were essentially the same as those of the first series except for employing 2 steps of partition chromatography instead of extensive gel‐filtration, gave about 0.1 mg of purified sample which served as material for studies of the amino acid composition and electrophoretic properties of GSS.

Starfish Gonad: Action and Chemical Identification of Spawning Inhibitor

The gamete-shedding substance obtained from the radial nerves induces spawning when it is applied to the gonads of mature starfish in vivo and in vitro. A substance that inhibits the action of this spawning factor is present in both ovary and testis; it has been isolated from testis of Asterina pectinifera and chemically identified as L-glutamic acid.

Increase in dATP pool in aphidicolin-resistant mutants of mouse FM3A cells.

Abstract Mutants that were resistant to aphidicolin were isolated from mutagenized mouse FM3A cells at a frequency of about 10−6. Resistance to aphidicolin in these mutants was not due to an effect on [3H]thymidine incorporation into DNA, DNA synthesis in permeabilized cells, or DNA polymerase α. All the mutants showed a greatly increased dATP pool and decreased ability to incorporate [3H]deoxycytidine into DNA. They also showed cross-resistance to both 1-β-D-arabinofuranosyladenine and 1-β-D-arabinofuranosylcytosine. These results indicate that an enzyme involved in production of dATP or its regulation is altered in these mutants. It is suggested that dATP competes with aphidocolin at its killing site or that dATP reverses the effect of aphidicolin by some unknown mechanism in vivo .

SELECTIVE INHIBITION BY APHIDICOLIN OF THE ACTIVITY OF DNA POLYMERASE ALPHA LEADS TO BLOCKADE OF DNA SYNTHESIS AND CELL DIVISION IN SEA URCHIN EMBRYOS

Aphidicolin at 2 μg/ml caused 90% inhibition of mitotic cell division of sea urchin embryos at the I‐cell stage. However, at 40 μg/ml it did not affect meiotic maturational divisions of starfish oocytes, which do not involve DNA replication. At 2 μg/ml it caused 90% inhibition of incorporation of tritiated thymidine into DNA of sea urchin embryos but did not affect protein or RNA synthesis even at a higher concentration. At 2 μg/ml it also caused 90% inhibition of the activity of DNA polymerase α, obtained from the nuclear fraction of sea urchin embryos, but did not affect the activity of DNA polymerase β or γ. These findings suggest that DNA polymerase α is responsible for replication of DNA in sea urchin embryos.

Characterization and action of meiotic maturation inhibitors in starfish ovary

Mechanical release of oocytes from the ovary of the starfish Asterias amurensis into sea water results in “spontaneous” meiotic maturation of the oocytes. The substances blocking the maturation of Asterias oocytes have been purified from the ovary and shown to be steroid glycosides named asterosaponins A and B. The extract prepared from isolated oocytes was incapable of inhibiting oocyte maturation. The ovarian extract inhibited the production of 1-methyladenine (1-MA) in follicle cells surrounding the oocyte. The ovarian extract failed to influence 1-MA-induced maturation of the oocyte with or without follicle cells. It can be concluded from the present results that the role of the ovarian extract containing steroid glycosides is to arrest “spontaneous” production of 1-MA in follicle cells. The suppression can be overcome by the action of a gonadotropic peptide hormone released from the nerve tissue.

Aphidicolin resistant mutant of which DNA polymerase α is induced by this drug

Abstract Mutants resistant to aphidicolin, a specific inhibitor of DNA polymerase α of eukaryotic cells, were selected from cultured FM3A cells, derived from mouse mammary carcinoma. One of them, designated as Aph 212, grew in the presence of 1 μg/ml of the drug, which did not permit wild type cells to grow. The resistance of Aph 212 cells to aphidicolin seems to be due to the increment of the activity of DNA polymerase α when Aph 212 cells were cultivated in the presence of the drug.

Studies on asterosaponins—V : A novel steroid conjugate, 5α-pregn-9(11)-ene-3β,6α-diol-20-one-3-sulfate, from a starfish saponin, asterosaponin A

Abstract A new crystalline steroid conjugate was obtained by partial acid hydrolysis of asterosaponin A, a steroidal saponin from the starfish, Asterias amurensis . The structure of the conjugate was established as 5α-pregn-9(11)-ene-3β,6α-diol-20-one-3-sulfate ( 2 ) on the basis of elemental analysis, IR and PMR spectra measurement and chemical reaction. Solvolysis of compound 2 yielded 5α-pregn-9(11)-ene-3β,6α-diol-20-one. Oxidation with chromium trioxide-pyridine complex followed by solvolysis afforded a new steroid, 5α-pregn-9(11)-ene-3β-ol-6,20-dione, whose structure was deduced by the measurement of ORD curve and PMR spectra. Thus, the location of carbohydrate moiety in asterosaponin A has been assigned to 6α-hydroxy group of the steroid conjugate.

Inhibition of DNA synthesis in the adenovirus DNA replication complex by aphidicolin and 2′,3′-dideoxythymidine triphosphate

Abstract DNA synthesis in the adenovirus DNA replication complex, containing host DNA polymerases-α and -γ, was inhibited completely by aphidicolin and by 2′,3′-dideoxythymidine triphosphate (ddTTP). Double reciprocal plots of DNA polymerase activity in the replication complex against each dNTP gave a straight line although the complex contained two species of DNA polymerase. Inhibition by aphidicolin of DNA polymerase activity was competitive with dTTP but that of purified DNA polymerase-α isolated from adenovirus infected KB cells was competitive with dCTP. The above results suggest that DNA polymerases-α and -γ are integrated in the replication complex to behave as a single enzyme.