Stress-induced reversal of microRNA repression and mRNA P-body localization in human cells.

In metazoa, microRNAs (miRNAs) imperfectly base-pair with the 3'-untranslated region (3'UTR) of mRNAs and prevent protein accumulation by either repressing translation or inducing mRNA degradation. Examples of specific mRNAs undergoing miRNA-mediated repression are numerous, but whether the repression is a reversible process remains largely unknown. Here, we show that cationic amino acid transporter 1 (CAT-1) mRNA and reporters bearing the CAT-1 3'UTR or its fragments can be relieved from the miRNA miR-122-induced inhibition in human hepatoma cells in response to different stress conditions. The derepression of CAT-1 mRNA is accompanied by its release from cytoplasmic processing bodies (P bodies) and its recruitment to polysomes, indicating that P bodies act as storage sites for mRNAs inhibited by miRNAs. The derepression requires binding of HuR, an AU-rich-element-binding ELAV family protein, to the 3'UTR of CAT-1 mRNA. We propose that proteins interacting with the 3'UTR will generally act as modifiers altering the potential of miRNAs to repress gene expression.

Detection of different viral strains of hepatitis B virus in chronically infected children after seroconversion from HBsAg to anti-HBs indicating viral persistence.

BACKGROUND/AIMS: Seroconversion to anti-HBs or the loss of HBsAg is usually associated with complete elimination of the replicative hepatitis B virus. Usually in these patients hepatitis B virus DNA (HBV DNA) becomes undetectable. Routine controls of patients who underwent anti-HBs seroconversion by more sensitive tests showed that in some cases the virus persisted in the patient. Therefore the aim of our study was to evaluate if virus persistence could also be found in children with chronic hepatitis B after anti-HBs seroconversion. The virus pool should be characterized before and after seroconversion. METHODS: Viral DNA was extracted from nine HBsAg negative or anti-HBs positive sera of children, previously diagnosed as chronic HBsAg carriers. HBV DNA was amplified by polymerase chain reaction. Subsequently the nucleotide sequences of the polymerase chain reaction product in the a-determinant region (aa 121-161) were analyzed on an automatic fluorescent sequencer. RESULTS: In the sera of seven children, HBV DNA was detected in the HBsAg negative phase of the HBV infection. Mutations in codons 122, 125, 127, 131, 134, 143, 159 and 161 of the S gene could be documented, resulting in amino acid changes. In three patients the sequence analysis revealed changes in the HBV genotype from genotype A (serotype adw) to genotype D (serotype ayw) during seroconversion to anti-HBs. CONCLUSIONS: These data demonstrate that persistence of the hepatitis B virus can also occur in HBsAg negative and anti-HBs positive children. After loss of HBsAg, no specific HBV variant was identified. Although a conclusive explanation for the selection process cannot be provided, it remains a fact that the 'surviving' viral strain was mostly represented by genotype D.

Long-term persistence of hepatitis B virus DNA in the serum of children with chronic hepatitis B after hepatitis B e antigen to antibody seroconversion.

To evaluate the prevalence and duration of viremia in relation to the features of liver disease, we investigated hepatitis B virus (HBV) DNA by the polymerase chain reaction in the serum of 39 children with chronic hepatitis B, after hepatitis B e antigen to antibody seroconversion. During a mean observation period of 8.2 +/- 3.8 years after seroconversion, all patients were asymptomatic; 36 had persistently normal alanine aminotransferase levels, and three had occasional mild alterations. Liver histology, checked in 21 patients, showed persistent hepatitis in nine, fibrosis in 10, and cirrhosis in two cases. HBV DNA was always undetectable by dot blot hybridization. Five children eventually cleared hepatitis B surface antigen, including one with cirrhosis who developed liver cancer at 19 years. HBV DNA was detected by polymerase chain reaction in 87% of children within 5 years of follow-up, in 58% of cases 6-10 years after seroconversion (p < 0.001), and in 50% of patients investigated later. Long-term viremia was found in two patients (40%) who cleared HBsAg, including the one who developed liver cancer. The chances of clearing viremia during follow-up were higher in children with acute hepatitis at the onset of illness (86%) than in those with asymptomatic onset (37%; p < 0.05). Our results show that low levels of HBV viremia, probably reflecting low levels of virus replication, persist for several years in children with chronic hepatitis B after hepatitis B e antigen to antibody seroconversion and remission of liver disease, even after the clearance of hepatitis B surface antigen. Persistent replication could support mild biochemical alterations and inflammatory liver lesions. It could allow late reactivation of liver disease and may play a role in the development of carcinoma.

Selection of a precore mutant after vertical transmission of different hepatitis B virus variants is correlated with fulminant hepatitis in infants.

The incidence of perinatal transmission of hepatitis B virus (HBV) depends on the HBeAg/anti-HBe status of the mother. While children of HBeAg-positive mothers have a 90% probability of acquiring a chronic hepatitis B virus carrier state, babies of anti-HBe-positive mothers are more likely to develop fulminant hepatitis within the first 3 to 4 months of life. There is evidence that precore (pre-C) mutations of the HBV can be associated with fulminant hepatitis. The pre-C region was therefore examined in sera from nine infants with fulminant hepatitis after vertical transmission, one HBeAg-positive and seven anti-HBe-positive mothers by polymerase chain reaction (PCR) and direct sequence analysis. In five mother/infant pairs the virus populations were characterized in addition by analysing clones of the amplified products. All mothers were infected with two or four variants of HBV with mutations at different positions of the preC genome including position 1896, which results in a stop codon. While the precore stop codon was detected in a portion of the virus populations of the HBeAg-positive and of four anti-HBe-positive mothers the dominating viral strain was represented by the wild type virus in three. In contrast, the virus populations of all babies showed the 1896 precore variant as the prevalent virus strain during the phase of active disease. In the surviving baby only wild type sequences were detected after recovery. Subtype ayw was found in all mothers and infants and adw2 was present in three mothers and in the surviving child. The findings suggest that all mothers carried a wild type HBV population with a certain number of different HBV variants. After transmission of the mixed virus population a selection process was started in the baby. The association of subtype ayw with the precore mutations and with the fatal outcome of the hepatitis B might be the result of a directed selection of this variant with a particular advantage in the viral life cycle.

K-region oxides and imines derived from alkylated benz[a]anthracene congeners: synthesis, stability in aqueous media and mutagenicity.

The K-region oxides and imines of benz[a]anthracene, 1-methylbenz[a]anthracene, 7-methylbenz[a]anthracene, 7-ethylbenz[a]anthracene and 7,12-dimethylbenz[a]anthracene were synthesized and characterized (melting point, 1H-NMR and electron impact mass spectra, elemental analysis, IR spectroscopy). All 10 compounds showed high mutagenic activity in Salmonella typhimurium (reversion of his- strains TA97, TA98, TA100 and TA104). The arene imines were more potent than the corresponding arene oxides. Alkyl substitutions strongly influenced the activities. Furthermore, all compounds were more active when exposure took place in the absence of inorganic ions than when KCl (125 mM) was present. The influence of the exposure medium was more pronounced with strain TA98 than with strain TA100. The half-lives of the test compounds were determined from mutagenicity experiments in which the compound was added to the exposure medium at varying times before the bacteria. In dilute sodium phosphate buffer (10 mM, pH 7.4), the half-lives of these chemicals (or their biological activity) varied from 0.5 to 110 min. Addition of KCl (150 mM) did not measurably affect the half-lives of some test compounds and appeared to slightly shorten those of others. Therefore, it is unlikely that the strong effect of KCl on mutagenicity and the dependence of this effect on the bacterial strain used can be explained by influences of KCl on the test compounds. Rather, it appears more likely than an effect of KCl on the bacteria may be an important factor. This study provides further examples of strong influences of unobtrusive media components on mutagenicity. It also demonstrates that small structural changes (alkyl substituents at diverse positions of the aromatic system) may play an important role in chemical reactivity and biological activity.

Strong influence of the exposure medium on mutagenicity in the Ames test: 7-methylbenz[a]anthracene-5,6-oxide.

We have previously shown that the activity of the ionized mutagen, 1-hydroxmethylpyrene sulphate, is strongly enhanced in Salmonella typhimurium TA98, when KCl is present in the exposure medium (50-fold at a concentration of 125 mM KCl) and that the halogen ion is responsible for this effect. Here we show that KCl has the opposite effect on the activity of the lipophilic mutagen, 7-methylbenz[a]anthracene-5,6-oxide, (10-fold decrease at a concentration of 125 mM) and that K+ accounts for this influence. Many other solutes also decreased the mutagenicity of 7-methylbenz[a]anthracene-5,6-oxide, but to a smaller extent than the K+ salts. The stability of 7-methylbenz[a]anthracene-5,6-oxide did not appear to be altered in the presence of KCl (t1/2 approximately 12 min), as determined from mutagenicity experiments in which the test compound was added to the exposure medium at varying times before the bacteria. Furthermore, the influence of the exposure medium was significantly stronger in strain TA98 than in strain TA100. Taken together these findings argue for an influence of the medium on the bacteria rather than on the test compound. Parallel studies with other mutagens indicate that exposure in distilled water enhances the mutagenicity of many compounds. Exposure in distilled water, in combination with some other modifications, led to a 400-fold increase of the assay sensitivity towards 7-methylbenz[a]anthracene-5,6-oxide, as compared to the usual plate incorporation assay.