Wayne E. Magee

Immunological evidence for the appearance of a new DNA-polymerase in cells infected with vaccinia virus☆

Abstract Rabbits were immunized with a partially purified DNA-polymerase from control HeLa cells. γ-Globulin was isolated from the antisera and shown to contain antibodies which inhibited the enzymatic activity. The anti-DNA-polymerase γ-globulin did not inactivate the new DNA-polymerase made in response to vaccinia virus infection. All the enzyme increase after infection was due to the new enzyme which could be detected in particulate fractions, or in solubilized form after high speed centrifugation or sucrose density gradient centrifugation. It was concluded that the DNA-polymerase induced by vaccinia virus infection is immunologically distinct from the normal host enzyme.

Absorption of prostaglandins by the intestine and vagina of the rat

Abstract An in situ method was used to determine the rate of absorption of prostaglandins★★ E2, F2α, 15-methyl F2α and 15-methyl F2α methyl ester from the small intestine of the rat. Prostaglandin E2 disappeared from the lumen with a t 1 2 of 37 min. From 2–3 % of the radioactivity of the dose was recovered in the blood at 30–60 min, but the compound was extensively metabolized, and intact prostaglandin E2 did not exceed 0.04–0.08% of the dose. Absorption of the more polar prostaglandin F2α and 15-methylprostaglandin F2α proceeded at a slower rate with a t 1 2 of 60–70 min. Radioactivity in the serum at 30–60 min did not exceed 2 % of the dose for those animals given prostaglandin F2α or 0.8% of the dose for those animals given 15-methylprostaglandin F2α. A rapid, initial disappearance of 15-methylprostaglandin F2α methyl ester from the small intestine was observed, followed by a second, much slower rate of absorption. The rapid phase of the absorption was extended at high dosages and resulted in blood levels of radioactivity up to 7 % of the dose when 40 mg/kg were given. These results correlated with the rate of hydrolysis of the methyl ester in the lumen of the intestine which was slower at higher doses. Little or no methyl ester was detected in serum or intestinal tissue, but up to 1 .8 % of the dose of intact 15-methylprostaglandin F2α was estimated to be in the serum at 30 min. Additional experiments showed that 15-methylprostaglandin F2α methyl ester also was absorbed through the large intestine and the vagina. In each case, 15-methylprostaglandin F2α was found in the blood. Metabolites consisted of more polar acids and a component which migrated close to 15-methylprostaglandin F2α on thinlyer chromatograms. The extent of metabolism of 15-methylprostaglandin F2α methyl ester by the liver was determined in a perfusion experiment. The methyl ester was hydrolyzed within 1 min, and the free acid was taken up almost quantitatively by the circulating prostaglandins E and F occurs via 15-dehydrogenation, our results indicate that the liver is capable of extensively degrading the compounds to more polar meta-boites. Further evidence on this point comes from a liver perfusion experiment carried out as described above but with [5,6-3H2]prostaglandin E1. By 60 min, over 50% of the radioactivity in plasma was recovered in volatile form 3H2O and 17% remained in the aqueous phase, indicating extensive carboxyl sidechain cleavage. (An additional 10.3 % chromatographed as prostaglandin E1). Dawson et al.29 have reached similar conclusions. These observations may explain the lack of any prolongation in cardiovascular responses in vivo by the 15-methyl analogs over that observed with the parent compounds17 but does not offer an explanation for the enhanced antifertility activity of the analogs in hamsters, monkeys and humans17,30–32. It would, of course, be possible to speculate that cardiovascular activity relies on continuing levels of compound in the circulation while duration of antifertility activity is controlled by the local action of 15-hydroxyprostaglandin dehydrogenase on prostaglandins at target tissues17. Uterine tissue was found to contain relatively little dehydrogenase33, but high levels have been reported in human placenta34. A number of orally active drugs have been found to have t 1 2 values between 8 and 50 min using the present technique18. Therefore, barriers to successful oral dosage forms for the prostaglandins do not lie in their rate of uptake, but rather in the extensive metabolism occurring during absorption. The extremely low amounts of intact prostaglandin E2 that survived intestinal transport, and the detection of a metabolite in the lumen of the intestine as well as in serum with the correct RF for the 15-keto compound suggest that the 15-dehydrogenation reaction poses the major metabolic block to successful intestinal absorption. In addition, dehydrogenase activity was readily detected in intestinal washes (unpublished observations). It is not possible to completely evaluate the relative role of the various degradative pathways in the intestine versus those in liver or lung during absorption on the basis of the present experiments. However, it is apparent that appreciable blood levels of intact drug could be obtained during intestinal absorption by employing an analog in which only the action of the dehydrogenase was blocked.

Inhibition by interferon of the uncoating of vaccinia virus

Abstract The effect of interferon was determined on those steps in vaccinia virus infection that precede release of viral DNA into the cytoplasm. These include loss of the outer protein coat of the particle which exposes the “core” and release of the DNA from the core (uncoating). Primary cultures of chicken embryo fibroblasts were treated with interferon and infected with radioactive vaccinia virus prepared with thymidine- 3 H in the DNA. At various times after infection the cells were ruptured, and the amounts of virus, cores, and viral DNA were determined after separation of these components by sucrose density centrifugation. Uncoating also was measured by DNase sensitivity of the viral DNA. Interferon did not alter either the rate of disappearance of virus particles or the rate of formation of cores. However, uncoating was inhibited strongly so that very little viral DNA was liberated and cores tended to accumulate. The response of uncoating to increasing concentrations of interferon was similar to that determined previously for the synthesis of viral DNA-polymerase and viral DNA. These observations suggest that uncoating is a viral function. Not enough cores accumulated in the interferon-treated cells to account for all the virus that disappeared. Experiments with heat-inactivated virus, and with normal virus in the presence of cycloheximide, showed that chicken embryo fibroblasts can digest both virus and cores to acid-soluble materials without accumulating acid-insoluble intermediates. The inhibition of uncoating obtained with cycloheximide closely resembled that seen with interferon.

An autoradiographic study on the intracellular development of vaccinia virus

Abstract The multiplication of intracellular vaccinia virus in HeLa cells was followed over the complete growth cycle by plaque assay after sonic disintegration of the infected monolayers. A constant proportion of the adsorbed virus lost identity as plaque-forming units immediately after infection. These particles were considered in eclipse since this loss persisted throughout the lag phase. Nuclear radioactivity of host cells prelabeled with thymidine- H 3 was not transferred appreciably to the cytoplasm upon infection, indicating that host deoxyribonucleic acid was not incorporated into the newly synthesized virus. Occasional bizarre transfer of nuclear label to the cytoplasm was observed. Infection stimulated an early nuclear response which was observed most readily with prelabeled cells. Thymidine- H 3 was incorporated into the cytoplasm of cells labeled after infection, and the sites of accumulation of label were considered to be the sites of viral DNA synthesis. Thymidine- H 3 accumulated most rapidly during the period between 1.5 and 6 hours after infection. The virus units migrated outward from the centers of synthesis after formation and dispersed throughout the cytoplasm. Inclusion bodies lost label when observed during the late hours following infection, and the radioactivity was found throughout the extensions of the cell.

Effect of interferon on early enzyme and viral DNA synthesis in vaccinia virus infection.

Abstract The quantities of infectious virus and viral DNA-polymerase produced and the rate of viral DNA synthesis were all inversely related to the interferon concentration in vaccinia virus-infected monolayers of whole chicken-embryo fibroblasts. However, virus production was a least 10 times more sensitive to interferon inhibition than were the other parameters studied. Increasing concentrations of interferon depressed the initial rate of synthesis of the DNA polymerase as well as its final yields. The response of the rate of DNA synthesis to interferon concentration was not affected by varying the virus inoculum from 0.1 to 4.0 plaque-forming units per cell. Radioautographic studies of viral DNA synthesis demonstrated that inhibition by interferon is not “all or none” even at the level of the vaccinia inclusion. Instead, increasing concentrations of interferon reduced the quantity of DNA synthesized per site in addition to the proportion of cells initiating DNA synthesis and the number of DNA synthesizing sites per cell. A large proportion of the cells initiated viral DNA synthesis even after treatment with an interferon concentration that reduced virus yield 98%.

THE EFFECTS OF INTERFERON ON VACCINIA VIRUS INFECTION IN TISSUE CULTURE

Virus-induced acid deoxyribonuclease (DNase) waa assayed by Method I1 of McAuslan and Kates’. Samples containing lo7 infected ce l l s were suspended in 0.3 ml of 0.01 M Tris buffer (pH 7.4) containing 0.55 deoxycholate and disrupted sonically by 3 min of treatment in a Raytheon sonicator. Then, 20 pl of the enzyme suspension was added to 20 p 1 of 70

BIOCHEMICAL STUDIES ON THE ANTIVIRAL ACTIVITIES OF THE ISATIN‐β‐THIOSEMICARBAZONES*

saturated ammonium sulfate, and to this mixture was added 0.35 a1 of 0.01 M acetate buffer (pH 4.8) containing 43 pg of H3-labeled, heat-denatured DNA. After incubation at 3’7°C for 30 min or l k hr, the liberated acid-soluble radioactivity was determined. Enzyme activity was expressed as counts per minute released per mg of protein, with the effect of interferon expressed as a percentage of the activity in untreated cells.

Intestinal absorption of prostaglandin F2α, 15(S)-15-methyl prostaglandin F2α and their methyl esters in the dog

Our earlier work (Bach & Magee, 1962) on the mechanism of action of isatin P-thiosemicarbazone (ITSC), as well as that of other investigators (Easterbrook, 1962; Appleyard et al., 1962), lead to the conclusion that the primary site of action of this compound lies somewhere in the late phases of virus replication. Thus, we found that concentrations of ITSC sufficient to inhibit virus replication did not influence the initial burst of thymidine-H3 incorporation into DNA by infected cells, and examination of the cells by microautoradiography ( Magee et al., 1959; Magee et a/., 1960; Kato et a/., 1960; Cairns, 1960) showed the apparently normal establishment of cytoplasmic centers of DNA accumulation. ITSC blocked virus production whenever it was added to infected cells. Moreover, a brief exposure to ITSC during the first few hours of infection also resulted in markedly decreased final virus yield. Viral inhibitory concentrations of ITSC did not prevent cytopathic changes in infected cells (Bach & Magee, 1962; Easterbrook, 1962; Appleyard et al., 1962); such cells did not survive and could not be cloned (Sagik & Wright, 1962). This “lethal” effect appeared to be due to the virus itself and the partial cycle of replication that it induced. Numerous studies have demonstrated that a complete virus cycle is not necessary for cytopathic changes in the cell. Vaccinia virus that was inactivated with heat or ultraviolet light caused cell death at high multiplicities of infection (Hanafusa, 196Oa & 19606). Appleyard et al. (1962) could find only three “early” antigenic lines in extracts from cells that were infected with ultraviolet treated virus using an immunodiffusion technique in agar, compared to some 15 lines that appeared during the course of normal infection. Thus, the toxic reaction may be caused by an “early” product of infection. The present paper seeks to shed some light on the events that ultimately cause cell death by comparing the actions of ITSC and some of its analogues with those of other inhibitors of vaccinia virus replication. A preliminary report of some of these data has appeared (Magee & Miller, 1964).

Treatment of Murine Cryptococcosis with Liposome-Associated Amphotericin B

Abstract Radioactive prostaglandins were introduced into intact intestinal loops of dogs, and the venous blood draining from the segments was collected and analyzed for absorbed drugs and metabolites. From 8 to 18% of initial radioactivity accumulated in the blood in 60 min after (9- 3 H]prostaglandin F 2α or its methyl ester were given. The compounds were extensively metabolized in the intestinal mucosa. Less than 0.3 % of the dose was recovered from the blood as intact prostaglandin F 2α when this compound was given, and up to 1.7% of the dose was found as prostaglandin F 2α following administration of prostaglandin F 2α methyl ester. A methyl group at carbon-15 of the prostaglandin molecule slowed down intestinal absorption considerably. From 0.8 to 1.2% of the radioactivity of the dose accumulated in the blood in 60 min when 14 C-labelled 15(S)-15-methylprostaglandin F 2α was given. The methyl ester was absorbed more readily, and 2.4–4.7% of the dose reached the blood. In both cases, 25–50% of plasma radioactivity appeared as free acid (blood levels of 0.3–4.2% of the dose). Esterase activity was detected in mucosa and plasma; therefore, very little ester appeared in the blood. We conclude that intestinal administration of 15(S)-15-methylprostaglandin F 2α results in appreciable blood levels of intact compound. A methyl group at carbon-15 blocks the action of prostaglandin dehydrogenase, without preventing other degradative reactions of prostaglandins.