Table 1. The radioactive product was sedimented through a neutral sucrose gradient and appeared as a single peak of about 13-14 S (Fig. 1). This S value corresponds to a molecular weight for linear double-stranded DNA of 1.8 to 2.3 X 106 (8).Aliquots of each fraction of the neutral sucrose gradient were digested with endo R-HaeIII and the fragments were separated on a 5% polyacrylamide gel (Fig. 1). The sizes of the HaeIII fragments indicated in Fig. 1 as nucleotide pairs were calculated from the migration positions relative to those of the polyoma HaeIII fragments run on the same gel (6, 9).Undigested aliquots of fractions 14-20 of the sucrose gradient in Fig. 1 were subjected to electiophoresis through a 2% polyacrylamide-0.5% agarose gel (Fig. 2). In vitro replicated DNA from Dane particles can be resolved into two Il of H20 and 2 Ml of 1 M NaOH was added to denature the DNA.After 5 min at 40, the sample was adjusted to pH 7.5 with 30 Ml of 0.33 M Tris.HCl, pH 7. Seventy Ml of H20 containing 0.7 Mmol of MgCl2, 1 nmol each dATP, dCTP, and dGTP, 0.5 nmol of [a-32P]_ dTTP (10 Ci/mmole), 2 Mg of oligonucleotide primers, and 0.5 jg of DNA polymerase I were added and the reaction was incubated at 15°. Aliquots were removed and assayed for incorporation of radioactive precursor into acid-insoluble material.
An aqueous extract of the plant PhyUanthus niruri inhibits endogenous DNA polymerase of hepatitis B virus and binds to the surface antigen of hepatitis B virus in vitro. The extract also inhibits woodchuck hepatitis virus (WHV) DNA polymerase and binds to the surface antigen of WHV in vitro. The extract, nontoxic to mice, was tested for antiviral activity in woodchucks (Marmota monax). In a trial using six long-term WHV-carrier woodchucks, five treated animals showed a faster decrease in woodchuck hepatitis virus surface antigen titer compared to one untreated control. In animals recently infected with WHV, the extract was effective when administered i.p. in three out of four animals in reducing and within 3-6 weeks eliminating both the surface antigen titer and DNA polymerase activity in serum. The treatment was discontinued after 10 weeks, and the treated animals have remained free of detectable markers of WHV for more than 45 weeks. In contrast, three untreated controls remained positive for both markers for WHV. One of the controls died after 8 weeks; the other two controls have remained positive for WHV markers for more than 45 weeks. In a third trial with long-term carriers, test animals treated subcutaneously with the extract for 12 weeks did not respond; but on switching the mode of administration to i.p., two out of the five animals showed a significant decrease in woodchuck hepatitis virus surface antigen titer compared to controls.Chronic carriers of the hepatitis B virus (HBV) may remain asymptomatic for long periods, but many are at high risk of eventually developing post-hepatitic cirrhosis and primary hepatocellular carcinoma. Carriers are often infected within the first few years of life, but symptoms of chronic liver disease and primary hepatocellular carcinoma may not be perceived until the third, fourth, or later decades; pathogenesis, even though relentless, is slow (1, 2).Materials of animal, bacterial, and plant origin (3) have been described that appeared to interfere with the binding of the HBV surface antigen (HBsAg) to the HBsAg antibody (anti-HBs). Subsequently, about 1200 species of plant were tested, and about one-third were found to inhibit antiHBs-HBsAg binding. To obtain more specificity and increase the probability of obtaining an effective therapeutic agent, in addition to the inhibition of HBsAg-anti-HBs binding, we examined plant extracts in vitro to determine if they inhibited the endogenous DNA polymerase (DNAp) of HBV, which is necessary for its replication. The first plant tested was Phyllanthus niruri, which has been and is used widely (4) To assess the effects of P. niruri on the replication of HBV-like viruses in vivo, we used the woodchuck (Marmota monax) as an animal model. The carrier state in woodchucks and humans is similar. Liver diseases including primary hepatocellular carcinoma induced by woodchuck hepatitis virus (WHV) in woodchucks are very similar to those induced by HBV in humans. WHV is similar to HBV (6, 7) with substantial immunological cross-re...
DuRING RECENT YEARS. there have been parallel developments in understanding, on the one hand, the pathogenesis of primary hepatic carcinoma (PHC, hepatoma) and, on the other, the biology of Australia antigen (hepatitis B surface antigen) and the infectious agent, hepatitis B xirus (HBV), to-hich it is intimatel1 related. Recently, the
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