Hideo Nakane

Baicalin, an inhibitor of HIV-1 production in vitro.

The flavonoid baicalin markedly inhibits replication of human immunodeficiency virus type 1 (HIV-1) in a concentration-dependent manner in normal peripheral blood mononuclear cells (PBMC) stimulated with phytohemagglutinin (PHA) in vitro. The effect was more pronounced when the cells were pretreated with baicalin. Furthermore, baicalin inhibits HIV-1 replication in PHA-stimulated PBMC from asymptomatic HIV-1-seropositive carriers. The 50% inhibitory concentration for HIV-1 replication was approximately 0.5 microg/ml. At the concentration of 2 microg/ml of baicalin, copy numbers of HIV-1 proviral DNA were approximately 50 times less than in untreated controls. In a cell-free infection system, baicalin inhibited the activity of HIV-1 reverse transcriptase (RT), but not the activity of human DNA polymerases alpha and gamma (DNA polymerase beta was slightly inhibited), suggesting that the anti-HIV-1 effect of baicalin may at least partly be due to inhibition of HIV-1 RT.

Inhibition of reverse transcriptase activity by a flavonoid compound, 5,6,7-trihydroxyflavone.

5,6,7-Trihydroxyflavone (baicalein) is a potent inhibitor of the activities of reverse transcriptases from murine leukemia viruses (MLV) (Rauscher and Moloney strains) and human immunodeficiency virus (HIV). Under the reaction conditions specified for each of the MLV- and HIV-reverse transcriptases, both enzyme activities were inhibited by more than 90% in the presence of 2 micrograms/ml baicalein. The mode of the inhibition by baicalein was competitive with respect to the template.primer, (rA)n.(dT)12-18, and noncompetitive to dTTP substrate. Ki value of baicalein for the MLV-reverse transcriptase was determined to be 0.37 microM.

Inhibition of HIV-reverse transcriptase activity by some phloroglucinol derivatives

Four phloroglucinol derivatives, named mallotophenone (5‐methylene‐bis‐2,6‐dihydroxy‐3‐methyl‐4‐methoxyacetophenone), mallotochromene (8‐acetyl‐5,7‐dihydroxy‐6‐(3‐acetyl‐2,4‐dihydroxy‐5‐methyl‐6‐methoxybenzyl)‐2,2‐dimethylchromene), mallotojaponin (3‐(3,3(dimethylallyl)S‐(3(acetyl‐2,4‐dihydroxy‐5‐methyl‐6‐methoxybenzyl)‐phloracetophenone) and mallotolerin (3‐(3‐methyl‐2‐hydroxybut‐3‐enyl)‐5‐(3‐acetyl‐2,4‐dihydroxy‐5‐methyl‐6‐methoxybenzyl)‐phloracetophenone), have been tested for their ability to inhibit the activity of human immunodeficiency virus (HIV)‐reverse transcriptase. Under the reaction conditions with (rA)n · (dT)12–18 as the template · primer, the enzyme activity was inhibited by approximately 70% in the presence of 10 μ/ml mallotochromene or mallotojaponin, whereas mallotophenone and mallotolerin were much less inhibitory to the enzyme. The enzyme activity was also inhibited, though to lesser extent, by these compounds under similar conditions with initiated MS‐2 phage RNA as the template · primer, The mode of inhibition was, as analyzed with mallotojaponin, competivite with respect to the template · primer, (rA)n · (dT)12–18, and non‐competitive with respect to the triphosphate substrate, dTTP, The K i value of mallotojaponin for HIV‐reverse transcriptase was determined to be 6.1 μM.

Establishment and characterization of a transplantable erythroblastic leukemia in C3H mice

A disease with the characteristics of an erythroblastic leukemia was induced by X-ray irradiation of 300 Rads in C3H mice. The leukemia is transplantable in syngeneic mice by i.v. injection of the spleen cells. The mice show almost pure erythroid cells of various differentiation stages in peripheral blood. The number of total nucleated cells in the peripheral blood increased, but hematocrit and platelet number decreased. Reverse transcriptase activities were measured in spleen and liver of the mice and the data suggested that the leukemia was not induced by retrovirus infection. This leukemia is distinguishable, in this respect, from diseases reported by Friend or Rauscher. The leukemia will offer a good animal model for the studies on non-viral leukemogenesis and disorders of erythropoiesis.

Stromal Cell-dependent Growth of Leukemic Cells from Murine Erythroblastic Leukemia

Transplantable erythroblastic leukemia was induced by 300‐rad irradiation of C3H mice. Conditions for in vitro growth of the leukemic cells were studied. None of interleukin‐3, granulocyte/macrophage colony‐stimulating factor and erythropoietin could support the growth of the cells in vitro. In contrast, the leukemic cells grew into a stroma‐dependent cell line, ELM‐D, in close contact with the stromal cell layer of 900‐rad‐irradiated long‐term bone marrow culture. A stroma‐independent cell line, termed ELM‐I‐1, was further established from the non‐adherent population in the co‐culture of the leukemic cells, ELM‐D, with stromal cells. Reverse transcriptase activity was not detectable in ELM‐D or ELM‐I‐1 cells. Studies on binding and cross‐linking of 125I‐erythropoietin showed that ELM‐I‐1 cells had erythropoietin receptors, and two major radiolabeled protein products with molecular weights of 120 kDa and 140 kDa were detected on sodium dodecyl sulfate/polyacrylamide gel electrophoresis under reducing conditions.

Induction of cellular dan polymerases in a Burkitt lymphoma-derived cell line by treatment with 5-iododeoxyuridine

Cellular DNA polymerases of a Burkitt lymphoma-derived cell line (P3HR-1) were found to be greatly induced by treatment of the cells with 5-iododeoxyuridine (IUdR) at a concentration which induces Epstein-Barr virus (EBV) early antigen (EA) expression. The activities of all the DNA Polymerases alpha, beta and gamma in P3HR-1 cells increased 7-9 fold by exposure of the cells to IUdR (25 micrograms/ml) for 3 days, while the EBV-coded DNA polymerase activity in the cell remained undetectable under the assay conditions employed. Under the same culture conditions with IUdR, EA-positive P3HR-1 cells increased to 16.6% which was much higher than that of the non-treated control cells (0.32%). On the other hand, another Burkitt lymphoma cell line, Raji, had very low incidence (1.27%) of EA induction by IUdR-treatment and the level of DNA polymerase activities remained almost unchanged. From these results it seems that the increase in DNA polymerase activity during the treatment of P3HR-1 cells with IUdR is closely related to high incidence of EA expression in these Burkitt lymphoma cells. Also, the finding has revealed yet unknown effect of IUdR on cultured cells and provides a useful tool to obtain a large quantity of the induced cellular DNA polymerases from the P3HR-1 and KB cells.