Heterogeneous HBV mutants coexist in Korean hepatitis B patients.

Although many hepatitis B virus (HBV) mutants have been found in all open reading frames since the precore defective mutant was initially reported, systematic investigations of diverse HBV mutant populations in hepatitis B patients have not been performed. Therefore, we examined whether heterogeneous mutant populations simultaneously exist in Korean hepatitis B patients. In order to detect hepatitis B virus mutants, we amplified a conserved core region and a surface antigen region of HBV DNA by PCR from sera of 27 Korean chronic hepatitis B patients, and then performed single strand conformational polymorphism analysis followed by DNA sequencing analysis. The results showed that heterogeneous HBV mutants in both regions were present in a single as well as in various hepatitis B patients. Sequence analysis revealed a defective interfering particle with missense mutation in the core region. We also found that two subtypes of adr and adw coexisted in a single patient. In addition, a point mutation causing a stop codon in the surface antigen region was observed. We are further analyzing the clinical implications of HBV mutants to identify their roles in the pathogenesis of chronic hepatic disorders induced by HBV.

Primers determine the sensitivity of PCR-mediated hepatitis B virus DNA detection and pretreatment of PCR mixture with 8-methoxypsoralen eliminates false-positive results.

Most methods for the diagnosis of hepatitis B virus (HBV) infection largely depend on viral DNA detection by polymerase chain reaction (PCR) or radioimmunological assay of viral antigens or antibodies. The quality assurance program recently established in Europe reported that PCR-mediated HBV DNA detection methods used in many laboratories produced a high rate of false-positive and false-negative results. Thus, we attempted to improve the conditions of current PCR methods for detection of HBV DNA. In the present study, we applied a recently developed method of releasing HBV DNA from virion by NaOH treatment of patient serum. Using four different primer sets specific to the HBV core region, we found that the sensitivity of first-round PCR can be improved by more than two orders of magnitude depending on the primers. The second round of PCR using nested primers was sensitive enough to detect up to 10(-6) pg of the HBV DNA, which is equivalent to approximately 3 copies of the HBV genome. Among the approximately 800 HBV-infected patient sera investigated in our laboratory, more than 60% of the tested samples gave positive results in the first-round PCR. The rate of positive results obtained using our experimental conditions is very high in comparison with other reports. The reamplification of the first-round PCR reaction mixture with the nested primers produced practically 100% positive results. For diagnosis of HBV infection, we routinely used 1 microliter of patient serum, which was found to be optimum in our laboratory. Surprisingly, from 20% of our positive results, even serum diluted to 1/100 (0.01 microliter) produced a stronger signal than 1 microliter. This observation suggests that direct PCR amplification of HBV DNA released from serum by NaOH treatment has to be compensated by other DNA detection methods for correct quantitation. In order to eliminate the false positive signal resulting from the carry-over due to massive screening of a large number of samples, PCR reaction mixture containing 8-methoxypsoralen was exposed to ultraviolet light prior to thermal cycle amplification. This exercise did not decrease the sensitivity of the detection method, but almost completely removed the false positive results caused by contaminated templates. We are in the process of improving PCR-mediated HBV DNA detection methods to attain more reliable and easily applicable methods.

Insulin has a limited effect on the cell cycle progression in 3T3 L1 fibroblasts.

Insulin has pleiotropic effects on the regulation of cellular growth, differentiation, and metabolism. The biochemical events ultimately leading to cell proliferation after insulin treatment have been demonstrated in detail by numerous research groups. However, depending on cell types, it has been shown that insulin has various effects on cell proliferation. Therefore, we attempted to more critically evaluate the effect of insulin on cell proliferation in 3T3 L1 fibroblasts. In this study, we investigated insulin's effect on cell proliferation by using [3H]thymidine incorporation, flow cytometry, and cell counting. In 3T3 L1 fibroblasts studied in 0.5% serum, insulin induced a two-fold increase in [3H]thymidine incorporation over at 48 h, and the maximal rate of DNA synthesis was observed during 8-12 h incubation. The flow cytometric analysis also showed that insulin increased the cell population in the S phase. After insulin treatment for 48 h, cell numbers increased approximately 45% in comparison with 0.5% serum control. Cell division was found to occur only once in 60 h after staining 3T3 L1 fibroblasts with carboxyfluorescein diacetate succinimidyl ester (CFSE). Taken together, this data indicates that insulin stimulated the transit from the G0/G1 to S phase, progressed the cell cycle through the G2/M phase, and increased the cell number. However, under our experimental conditions, cells divided only once in 60 h in the presence of insulin.