Free flavins accelerate release of ferrous iron from iron storage proteins by both free flavin-dependent and -independent ferric reductases in Escherichia coli.

Ferredoxin NADP+ oxidoreductase (Fpr) and oxygen-insensitive NAD(P)H nitroreductase (NfnB) are purified from Escherichia coli JM109 (E. coli JM109) as a predominant free flavin-independent ferric reductase. In the present study, we prepared natural iron storage proteins, E. coli ferritin A (FtnA) and bacterioferritin (Bfr), to show the effective ferrous iron release from these proteins by Fpr and NfnB in the presence of free flavins. Fpr and NfnB showed flavin reductase activity for flavin adenine dinucleotide (FAD), flavin mononucleotide (FMN) and riboflavin, and their ferrous iron release activities were positively associated with the catalytic efficiencies (kcat/Km) for individual flavins. The ferrous iron release activity of E. coli cell-free extracts was affected by flavin reductase activity of the extracts. The Butyl TOYOPEARL column chromatography of the extracts, on the basis of NAD(P)H-dependent flavin reductase activity, resulted in the separation of six active fractions containing Fpr, NfnB, NAD(P)H-quinone oxidoreductase (QOR), flavin reductase (Fre) or alkyl hydroperoxide reductase subunit F (AhpF) as major components. Like Fpr and NfnB, recombinant QOR, Fre, and AhpF showed flavin reductase activity and ferrous iron release activity in the presence of free flavins, indicating an association of flavin reductase activity with ferrous iron releasing activity. Taken together, both free flavin-dependent and free flavin-independent ferric reductases in E. coli require free flavins to mediate an electron transfer from NAD(P)H to ferric iron in the iron storage proteins for the effective ferrous iron release.

Genetic heterogeneity of the precore and the core promoter region of genotype C hepatitis B virus during lamivudine therapy.

It has been reported that spontaneous or interferon (IFN)-induced hepatitis B e (HBe) seroconversion has usually been associated with the development of a stop codon in the precore region. However, the difference between lamivudine-induced seroconversion and spontaneous or IFN-induced seroconversion is not known. The aim of this study was to investigate the correlation between the evolution of the precore and core promoter mutations and lamivudine-induced seroconversion. Forty-five patients with chronic hepatitis B virus (HBV) infection who were treated with lamivudine for more than 1 year were enrolled. The nucleotide sequence of the precore and core promoter region was determined before and after treatment with lamivudine for 1 year. Among 29 patients who were hepatitis B e antigen (HBeAg)-positive before treatment, 12 (41.3%) lost HBeAg during the course of treatment for 1 year. Of these, eight patients (66.7%) still had precore wild type HBV after 1 year. After 1 year, reversion to precore wild type HBV was detected in 11 (64.7%) of 17 patients who had precore mutant HBV before treatment. Twelve (70.6%) of 17 patients who were persistently HBeAg-positive had precore wild type HBV before and after treatment for 1 year. Despite the loss of HBeAg, two thirds of the patients still had precore wild type HBV after the 1-year treatment. It is suggested that lamivudine-induced seroconversion differs from spontaneous or IFN-induced seroconversion in the change of nucleotides in the precore region. The reversion in the precore region may be caused by the difference of drug-susceptibility to lamivudine. The antiviral effect of lamivudine may be more effective in the precore mutant HBV than in the precore wild type HBV.

Severe alcoholic hepatitis successfully treated by leukocytapheresis: a case report.

BACKGROUND: The prognosis of severe alcoholic hepatitis is poor, and there is no established method for a cure. METHODS: A 34-year-old man was admitted to Kurume University Hospital because of severe liver dysfunction due to excess alcohol intake. He was treated with prednisolone and two sessions of granulocyte and monocyte adsorption apheresis (GCAP) using an Adacolumn, which removes leukocytes--especially granulocytes and monocytes--from the peripheral blood. We evaluated the changes in the serum levels of interleukin-6, interleukin-8, tumor necrosis factor-alpha, and soluble intercellular adhesion molecule-1, as well as the conventional liver tests and peripheral white blood cell count. RESULTS: Prednisolone was effective in the short term but resulted in an increase in C-reactive protein (CRP), peripheral leukocytes, and serum total bilirubin. GCAP performed on the 34th and 41st hospital days produced decreases in the white blood cell count, total bilirubin, and intercellular adhesion molecule-1. The patient survived, despite the expected poor prognosis on admission. CONCLUSIONS: GCAP is recommended as a potential therapeutic option for severe alcoholic hepatitis.

A real-time quantitative polymerase chain reaction method for hepatitis B virus in patients with chronic hepatitis B treated with lamivudine.

OBJECTIVES: During treatment of chronic hepatitis B with lamivudine, changes in the level of hepatitis B virus (HBV) DNA were investigated using a real-time polymerase chain reaction (PCR) method with a detection limit of 1.7 log copies/ml (50 copies/ml) to clarify its clinical significance, particularly the association between HBV DNA levels and the emergence of tyrosine-methionine-aspartate-aspartate (YMDD) mutants. METHODS: Twenty-four patients who had received lamivudine therapy for >1 yr were studied. HBV DNA levels were determined using transcription-mediated amplification for sera with >3.7 log genome equivalents/ml, the Roche Monitor kit for sera with >/=2.6 log copies/ml, and real-time PCR for sera with < 2.6 log copies/ml (the detection limit was 1.7 log copies/ml). Patients were classified into three groups according to the minimal HBV DNA level attained during lamivudine therapy: the <1.7 log copies/ml group (eight patients), the 1.7-2.5 log copies/ml group (five patients), and the >/=2.6 log copies/ml group (11 patients). RESULTS: Pretreatment HBV DNA levels were significantly lower in the <1.7 copies/ml group than in the other two groups (p < 0.05). Neither the emergence of YMDD mutants nor a virological breakthrough of serum HBV DNA was observed in any of the eight patients in the <1.7 copies/ml group. In contrast, in the 1.7-2.5 copies/ml and >/=2.6 copies/ml groups, virological breakthroughs resulting from the emergence of YMDD mutants were observed in two of five patients and in all 11 patients, respectively (p < 0.001). Virological breakthroughs were observed at a mean of 49.6 +/- 18.4 wk in 11 the patients in the >/=2.6 copies/ml group and at wk 107 and 115 in two patients in the 1.7-2.5 copies/ml group. CONCLUSIONS: The real-time PCR method is useful for predicting the emergence of YMDD mutants and the estimated time of their emergence.

Predictive factors for remission and death in 73 patients with autoimmune hepatitis in Japan.

The prognosis of patients with autoimmune hepatitis (AIH) has not been clearly defined. The aim of this study was to define the prognostic factors of AIH in a population with long-term follow-up in Japan. Seventy-three patients who were diagnosed as having type 1 AIH between January, 1972 - August, 1999 were enrolled in this study. Initial treatment included prednisolone (PSL) (n=62), other drug regimens (n=7), and none (n=4). We examined the relation between several factors obtained at diagnosis in relation to disease activity found at the final observation point (January, 2000 - April, 2000). Multivariate logistic regression and Cox regression were used for statistical analysis. During the observation period, 8 patients died of the following: hepatic failure (n=4), hepatocellular carcinoma (n=1), severe infection (n=1), and unknown causes (n=2). At the end point, the number of patients in complete remission was 13, those with a normal alanine aminotransferase (ALT) level requiring some treatment was 35, and those with an abnormal ALT level despite medication was 17. Factors related to remission were total bilirubin (TB) (Odds ratio, 0.87), and immunoglobulin G (IgG) (Odds ratio, 1.00). Factors related to death were the aspartate aminotransaminase (AST)/ALT ratio (Odds ratio, 11.67) and response to initial PSL regimen (Odds ratio, 0.03). The results of this study show an importance of achieving a good PSL response at onset, and that initial TB, the AST/ALT ratio, and IgG levels are useful for therapeutic strategy.

Interferon-gamma brings additive anti-viral environment when combined with interferon-alpha in patients with chronic hepatitis C.

T-cell hyporesponsiveness may lead to chronicity of hepatitis C virus (HCV) infection. We evaluated whether interferon (IFN)-gamma injection can bring a Th1-dominant environment to patients with chronic hepatitis C. Seventeen patients with genotype 1b received natural IFN-alpha 5MU daily for the first 2 weeks and three times a week for the next 22 weeks followed by natural IFN-gamma 1 MU daily for 2 weeks. In 4 of 17 patients (23.5%), alanine aminotransferase (ALT) was normalized and 3 of these 4 patients (75.0%) cleared HCV RNA. beta2 microglobulin (BMG), neopterin and soluble (s) Fas increased with IFN-alpha and increased more with IFN-gamma. Serum interleukin (IL)-12, CD4 and CD8 remained unchanged with IFN-alpha but increased after IFN-alpha was replaced by IFN-gamma. IL-10 was not changed either with IFN-alpha or gamma. Productions of IL-2, IFN-gamma and tumor necrosis factor (TNF)-alpha by peripheral blood mononuclear cells did not change by IFN-alpha therapy, however, they were enhanced at the end of IFN-gamma therapy. Productions of IL-2 and 4 were unaffected. These results show that some immune parameters become Th1-dominant by additional IFN-gamma in patients with chronic hepatitis C. Combination of these two IFNs should be explored.