Emergence of lamivudine-resistant variants, with amino acid substitutions in the Tyr-Met-Asp-Asp (YMDD) motif of hepatitis B virus (HBV) reverse transcriptase, is a serious problem in antiviral therapy. Presence of YMDD motif variants in patients who had never been treated with lamivudine has been reported recently. However, no analysis of nucleotide and amino acid sequences of these variants has been performed. In the present study, using polymerase chain reaction (PCR) with peptide nucleic acid (PNA) clamping, we detected many new variants, such as Tyr-Arg-Asp-Asp (YRDD), Tyr-Met-Asp-Asn (YMDN). Many of them had stop codon(s) in overlapping HBs gene. Although the biological activity of these HBV polymerase variants remains to be determined, our results showed that numerous quasispecies are created during virus replication. A typical lamivudine-resistant Tyr-Val-Asp-Asp (YVDD) variant was detected in only one of 62 (1.6%) anti-HBe patients with HBV infection before administration of lamivudine. This variant did not have the L528M mutation, which is often associated with YVDD variants, and lamivudine therapy in this patient suppressed HBV replication. Thus, care should be taken when interpreting the results of detection of YMDD variants, especially when the sensitivity of the assay is very high. Amplification of rare variants by PCR with PNA seems a useful tool to examine the emergence of drug-resistant variants as well as naturally occurring mutants, such as the hepatitis B e antigen (HBeAg) stop codon and vaccine escape mutants. Examination of rare variants should enhance the understanding of the mechanism for emergence of drug-resistant HBV variants and help in developing strategies for new antiviral drugs.
mel-18 is a mammalian Polycomb group gene encoding a transcriptional repressor with tumor suppressive activity. Overexpression of mel-18 in mice results in cell cycle arrest of B cells upon B cell receptor stimulation with downregulation of c-myc. This phenotype is rescued in mel-18/c-myc double-transgenic mice, suggesting that c-myc locates downstream of mel-18. In mel-18 transgenic mice, the downregulation of cyclins D2 and E; CDK4, -6, and -7; and CDC25A causes the impairment in the activities of cyclin-dependent kinases, resulting in hypophosphorylation of the retinoblastoma protein. In contrast, the upregulation of c-Myc, CDC25, and CDC2/CDK2 kinase activities results in the augmentation of B cell proliferation in mel-18-deficient mice. We therefore propose that mel-18 negatively regulates the cell cycle through a c-myc/cdc25 cascade.
SUMMARYWe examined development of autoimmune hepatitis in neonatally thymectomized C3H/HeN mice and tried to characterize the nature of liver antigens recognized by the autoantibodies at the molecular level. Autoantibodies to crude liver proteins detected by ELISA were found in 12 (67%) of 18 mice thymectomized 2 days after birth. However, autoantibodies were not detected in mice thymectomized 7 days after birth. The autoantibodies mainly consisted of IgG and reached the maximum level 8 weeks after birth. Hepatic inflammation, mononuclear cell infiltration in the portal area, was seen in 5 (28%) of 18 mice thymectomized 2 days after birth, but not in mice thymectomized 7 days after birth. Most infiltrating cells were Thy-1 lymphocytes. The serum autoantibody level to crude liver proteins in mice with hepatitis was much higher than that in mice without hepatitis. We fractionated crude liver proteins by a Sepharose 6B column and examined the reactivity against the autoantibodies. The autoantibodies of three of five mice with hepatitis reacted with the 150 kD liver proteins other than liver-specific protein (LSP). By Western immunoblotting of SDS-PAGE using LSP and fractionated liver proteins, we found that the molecular weights of the target antigens were 52 kD in LSP and 150 kD (strong band), 138, 128, 120 and 110 kD (weak band) in fractionated liver proteins other than LSP. This 150-kD target molecule in crude liver proteins was found only in liver. These results indicate that hepatitis and autoantibodies to liver proteins are induced spontaneously by neonatal thymectomy in mice, and the candidates of autoantigen in this hepatitis model are 52-kD protein in LSP and 150-kD liver proteins different from LSP. Still more, we regard the 150-kD molecule as a new autoantigen related to hepatitis.
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