Soft X-ray and ENA Imaging of the Earth's Dayside Magnetosphere.

The LEXI and SMILE missions will provide soft X-ray images of the Earth's magnetosheath and cusps after their anticipated launch in 2023 and 2024, respectively. The IBEX mission showed the potential of an Energetic Neutral Atom (ENA) instrument to image dayside magnetosheath and cusps, albeit over the long hours required to raster an image with a single pixel imager. Thus, it is timely to discuss the two imaging techniques and relevant science topics. We simulate soft X-ray and low-ENA images that might be observed by a virtual spacecraft during two interesting solar wind scenarios: a southward turning of the interplanetary magnetic field and a sudden enhancement of the solar wind dynamic pressure. We employ the OpenGGCM global magnetohydrodynamics model and a simple exospheric neutral density model for these calculations. Both the magnetosheath and the cusps generate strong soft X-rays and ENA signals that can be used to extract the locations and motions of the bow shock and magnetopause. Magnetopause erosion corresponds closely to the enhancement of dayside reconnection rate obtained from the OpenGGCM model, indicating that images can be used to understand global-scale magnetopause reconnection. When dayside imagers are installed with high-ENA inner-magnetosphere and FUV/UV aurora imagers, we can trace the solar wind energy flow from the bow shock to the magnetosphere and then to the ionosphere in a self-standing manner without relying upon other observatories. Soft X-ray and/or ENA imagers can also unveil the dayside exosphere density structure and its response to space weather.

BPR1J-097, a novel FLT3 kinase inhibitor, exerts potent inhibitory activity against AML.

BACKGROUND: Activating mutations of Fms-like tyrosine kinase 3 (FLT3) constitute a major driver in the pathogenesis of acute myeloid leukaemia (AML). Hence, pharmacological inhibitors of FLT3 are of therapeutic interest for AML. METHODS: The effects of inhibition of FLT3 activity by a novel potent FLT3 inhibitor, BPR1J-097, were investigated using in vitro and in vivo assays. RESULTS: The 50% inhibitory concentration (IC(50)) of BPR1J-097 required to inhibit FLT3 kinase activity ranged from 1 to 10 nM, and the 50% growth inhibition concentrations (GC(50)s) were 21±7 and 46±14 nM for MOLM-13 and MV4-11 cells, respectively. BPR1J-097 inhibited FLT3/signal transducer and activator of transcription 5 phosphorylation and triggered apoptosis in FLT3-driven AML cells. BPR1J-097 also showed favourable pharmacokinetic property and pronounced dose-dependent tumour growth inhibition and regression in FLT3-driven AML murine xenograft models. CONCLUSION: These results indicate that BPR1J-097 is a novel small molecule FLT-3 inhibitor with promising in vivo anti-tumour activities and suggest that BPR1J-097 may be further developed in preclinical and clinical studies as therapeutics in AML treatments.

Viral hepatitis-associated intrahepatic cholangiocarcinoma shares common disease processes with hepatocellular carcinoma.

Bile duct cells and hepatocytes differentiate from the same hepatic progenitor cells. To investigate the possible association of viral hepatitis B and C with intrahepatic cholangiocarcinoma (ICC), we conducted a retrospective case-control study using univariate and multivariate logistic analyses to identify risk factors for ICC. Besides hepatic lithiasis (25.6%; P<0.001), seropositivity for hepatitis B surface antigen (37.5% of all ICC patients; odds ratio (OR) =4.985, P<0.001) and seropositivity for hepatitis C antibodies (13.1%; OR=2.709; P=0.021) are the primary independent risk factors for ICC. Cirrhosis exerted synergic effects on the development of ICC. We compared the age distributions of viral-hepatitis associated ICC to that of viral hepatitis-associated hepatocellular carcinoma (HCC). The mean age of ICC patients with viral hepatitis B (56.4+/-11.1 years) were 9 years younger than that of ICC patients with viral hepatitis C (65.6+/-9.17 years), similar to that observed in HCC. The incidence ratio of HCC : ICC : CHC (combined hepatocellular cholangiocarcinoma) in our population was 233 : 17 : 1 consistent with the theoretic ratio of hepatocyte number to cholangiocyte number in the liver. Our findings indicated that both viral hepatitis-associated ICC and HCC shared common disease process for carcinogenesis and, possibly, both arose from the hepatic progenitor cells.

Acute leukaemia in chronic hepatitis B patients with lamivudine therapy.

Extensive clinical data have shown that lamivudine is an effective and safe drug for patients with chronic hepatitis B virus infection. No significant serious side effect has been reported. Four hundred and forty-eight patients with chronic hepatitis B, treated with lamivudine for more than 6 months, were closely monitored. Two patients developed acute myeloid leukaemia during or after lamivudine therapy. The first case developed acute myeloid leukaemia, 1 year after stopping lamivudine therapy, when A529T mutant HBV-DNA was still detectable. The second case achieved complete virological response but suffered from acute myeloid leukaemia during the ninth month of lamivudine treatment. D553N mutant hepatitis B virus was detected in granulocytes of her peripheral blood. Based on our lamivudine therapy data, the calculated incidence of acute myeloid leukaemia in patients during or after lamivudine therapy was higher in males and females than that of the general population. Whether lamivudine-selected viral mutations have enhanced activity/production of transcriptional transactivator and thereby increased the chance of leukaemic transformation of haematopoietic progenitor cells deserves further investigation.

Aberrant caspase-activated DNase (CAD) transcripts in human hepatoma cells.

The gene of caspase-activated DNase (CAD), the key enzyme for nucleosome cleavage during apoptosis, is mapped at chromosome 1p36, a region usually associated with hemizygous deletions in human cancers, particularly in hepatoma (HCC). It is tempting to speculate that CAD plays a tumour-suppressive role in hepatocarcinogenesis. To address this, we examined the CAD transcripts in six human HCC cell lines, one liver tissue from a non-HCC subject, and peripheral blood leukocytes (PBL) from three healthy individuals. Alternatively spliced CAD transcripts with fusion of exon 1 to exon 7 were isolated in most of the examined samples including HCC cells and normal controls. However, relatively abundant alternatively spliced CAD transcripts with fusion of exon 2 to exon 6 or 7, in which the corresponding domain directing CAD interaction with ICAD was preserved, were found only in poorly differentiated Mahlavu and SK-Hep1 cells. Interestingly, an abnormal CAD transcript with its exon 3 replaced by a truncated transposable Alu repeat was isolated in Hep3B cells, indicative of the implication of an Alu-mediated genomic mutation. Moreover, mis-sense mutations in the CAD genes were identified in all six HCC cell lines. Upon UV-induced apoptosis, DNA fragmentation efficiency was found to be intact, partially reduced and remarkably reduced in Huh7 and J328, Hep3B and HepG2, and Mahlavu cells, respectively. That mutations and aberrantly spliced transcripts for the CAD gene are frequently present in human HCC cells, especially in poorly differentiated HCC cells, suggests a significant role of CAD in human hepatocarcinogenesis.

Genome-wide hypomethylation in hepatocellular carcinogenesis.

Aberrant genome-wide hypomethylation has been thought to be related to tumorigenesis. However, its mechanism and implications in hepatocellular carcinogenesis remain to be elucidated. Samples of hepatoma (hepatocellular carcinoma, HCC) and paired non-HCC liver tissues were obtained from 17 HCC patients. Normal liver tissues obtained from three individuals were used as controls. Compared with the paired non-HCC liver tissues, genome-wide 5-methylcytosine content in HCC was reduced in all of the tested HCC samples (P < 0.001). Conversely, genome-wide 5-methylcytosine content did not significantly differ among normal, noncirrhotic, and cirrhotic liver tissues. Moreover, the degree of reduced DNA methylation was related to late histopathological HCC grade (P = 0.005) and large tumor size (P = 0.079). Compared with the paired non-HCC liver tissues, expression of DNA methyltransferases DNMT-1, DNMT-3A, and DNMT-3B and the DNA methyltransferase-like gene, DNMT-2, was up-regulated in 53, 41, 59, and 47% of the HCC samples, respectively. Surprisingly, small amounts of LINE-1 retrotransposon transcripts were detected in HCC and non-HCC as well as normal liver tissues, and the expression levels were not significantly different in HCC compared with the paired non-HCC or normal liver tissues. Of interest, the 3' ends of these LINE-1 transcripts were truncated. Our findings suggest that genome-wide hypomethylation in HCC is a continuing process that persists throughout the lifetime of the tumor cells rather than a historical event occurring in precancer stages or in cell origins for HCC. Up-regulation of DNA methyltransferases might simply be a result of increased cell proliferation in cancer. In addition, our results did not support the hypothesis of activation of transposable elements in HCC via genome-wide hypomethylation.

Correlation of mutational analysis to clinical features in Taiwanese patients with Gilbert's syndrome.

OBJECTIVES: Mutations in the promoter as well as in the coding region of the bilirubin UDP-glucuronosyltransferase gene (UGT1A1) have been found to be associated with Gilbert's syndrome. However, the genetic basis of Gilbert's syndrome in our population and correlation of these mutations to fasting serum bilirubin levels in patients with Gilbert's syndrome remain to be clarified. METHODS: We applied polymerase chain reaction-based direct-sequencing assays to examine mutations in UGT1A1 gene in 20 unrelated Gilbert's patients and in a family with Gilbert's syndrome. RESULTS: We studied three mutations that were previously reported to be associated with Gilbert's syndrome (i.e., the TATAA-box mutation, Gly71Arg, and Pro229Gln) in 20 patients with Gilbert's syndrome. Of the patients, 16, five, and six were found to have the TATAA-box, Gly71Arg and Pro229Gln mutations, respectively. Seven patients had simultaneous mutations both in the TATAA box and in the coding region. Of note, all six patients with Pro229Gln also had the TATAA-box mutation. Localization of Pro229Gln on the allele containing the TATAA-box mutation was demonstrated in a family with Gilbert's syndrome. The patients simultaneously heterozygous for both the TATAA-box mutation and Gly71Arg usually had serum bilirubin levels similar to those found in the patients homozygous for the TATAA-box mutation, but usually higher than those found in the patients heterozygous for the TATAA-box mutation alone. On the other hand, concurrence of Pro229Gln in patients with TATAA-box mutation or with Gly71Arg did not significantly affect serum bilirubin levels. CONCLUSIONS: The TATAA-box mutation and Gly71Arg are the major causes for Gilbert's syndrome in our population. Concurrence of mutations of Gly71Arg and TATAA-box usually exerts a synergistic effect on hyperbilirubinemia. Pro229Gln, which is regularly linked to the TATAA-box mutation, may not have a significant effect on serum bilirubin levels.

Olone modulates the therapeutic effect of interferon to eliminate preferentially the hepatitis B virus precore stop mutant.

BACKGROUND/AIMS: The aim of this study was to understand the changes in the proportion of hepatitis B virus precore stop mutant during the course of prednisolone primed interferon (IFN) therapy. METHODS: Three groups of patients were included: patients receiving prednisolone-primed IFN treatment (Group I, n=31), IFN treatment only (Group II, n=29), and placebo (Group III, n=25). The proportion of precore stop mutant was measured by a quantitative amplification-created restriction site method. RESULTS: Distinct patterns of the progression of the proportion of mutant were found among these three groups. A steady increase in the proportion of mutant was observed only in Group III patients. In Group II patients, the presence of a higher percentage of mutant (> 25%) immediately before IFN treatment was predictive for the subsequent clearance of hepatitis B e antigen (HBeAg) (p<0.01), but not for complete anti-viral response (p>0.05). Prednisolone pretreatment resulted in an increase in the proportion of mutant in patients with initially low percentages (< or = 25%) of mutant. During the period of IFN treatment, both the relative and absolute amount of the precore stop mutant decreased significantly in Group I patients who cleared HBeAg. The presence of such a decrease in this group of patients was predictive for both HBeAg clearance and complete anti-viral response. CONCLUSIONS: Our data suggest that prednisolone serves as a modulator to enhance elimination of precore stop mutant by IFN, which advocates the benefit of corticosteroid pretreatment in an area where the precore mutants are prevalent.

Identity of a novel swine hepatitis E virus in Taiwan forming a monophyletic group with Taiwan isolates of human hepatitis E virus.

Recently, we found that more than 10% of the cases of acute non-A, non-B, non-C hepatitis in Taiwan were caused by a novel strain of hepatitis E virus (HEV). Since none of these patients had a history of travel to areas where HEV is endemic, the source of transmission remains unclear. The recent discovery of a swine HEV in herd pigs in the United States has led us to speculate that HEV may also circulate in herd pigs in Taiwan and may serve as a reservoir for HEV in Taiwan. Of 275 herd pigs obtained from 10 pig farms in Taiwan, 102 (37%) were seropositive for serum anti-HEV immunoglobulin G (IgG). A 185-bp genomic sequence within the ORF-2 of the HEV genome was amplified and cloned from serum samples of an anti-HEV positive pig and subsequently from serum samples of a patient with acute hepatitis E. Sequence comparison revealed that the swine and human isolates of HEV share 97.3% identity. Phylogenetic analyses further showed that the Taiwan swine and human isolates of HEV form a distinct branch divergent from all other known strains of HEV, including the U.S. swine strain. To examine the potential risk of cross-species transmission of swine HEV to humans, the seroprevalences of anti-HEV IgG in 30 swine handlers, 20 pork dealers, and 50 control subjects were assessed and were found to be 26.7, 15, and 8%, respectively (for swine handlers versus controls, P = 0.048). Our findings may help provide an understanding of the modes of HEV transmission and may also raise potential public health concerns for HEV zoonosis.

Etiology of sporadic acute viral hepatitis in Taiwan: the role of hepatitis C virus, hepatitis E virus and GB virus-C/hepatitis G virus in an endemic area of hepatitis A and B.

The etiology of sporadic acute hepatitis was studied in 334 consecutive patients from Taiwan (237 men and 97 women, aged 16-81 years), with emphasis on the role of hepatitis C virus (HCV), hepatitis E virus (HEV), and GB virus-C/hepatitis G virus (GBV-C/HGV) in acute non-A, non-B (NANB) hepatitis and in HBsAg carriers with superimposed acute hepatitis. According to the conventional diagnostic criteria, there were 12 cases (3.6%) of acute hepatitis A, 17 cases (5.1%) of acute hepatitis B, 128 cases (38.3%) of acute NANB hepatitis, and 177 cases (53.0%) of acute hepatitis in HBsAg carriers (those who were HBsAg positive but IgM anti-HBc negative). Among 128 cases of acute NANB hepatitis, 70 (54.7%) had acute hepatitis C (HCV RNA positive), 5 (3.9%) had acute hepatitis E (IgM anti-HEV positive), and the other 53 (41.4%) were presumably acute hepatitis non-A-E. The prevalence of acute hepatitis A, B, E, and non-A-E showed no significant sex difference, whereas acute hepatitis C was significantly more prevalent in females. The prevalence of acute hepatitis A and B decreased and that of acute hepatitis C increased significantly with increasing age. In contrast, acute hepatitis E and non-A-E showed no significant age predominance. Of 177 HBsAg carriers with acute hepatitis, 64 (36.1%) demonstrated non-B hepatotropic virus superinfection, with HCV being the most common (60.9%), followed by hepatitis D, E, and A viruses, and the other 55 (31.1%) and 58 (32.8%) were presumed to have acute exacerbation of chronic hepatitis B or superimposed acute hepatitis non-A-E, respectively. Serum GBV-C/HGV RNA was detected in 3-4% of acute hepatitis non-A-E cases, suggesting its limited role in these cases.

High prevalence of TT virus infection in healthy children and adults and in patients with liver disease in Taiwan.

A newly identified DNA virus, named TT virus (TTV), was found to be related to transfusion-associated hepatitis. We conducted the following experiments to evaluate its pathogenic role in liver disease and potential modes of transmission. We used PCR to detect TTV DNA in serum. The rates of TTV viremia in 13 patients with idiopathic acute hepatitis, 14 patients with idiopathic fulminant hepatitis, 22 patients with chronic hepatitis, and 19 patients with cirrhosis of the liver were 46, 64, 55, and 63%, respectively, and were not significantly different from those in 50 healthy control subjects (53%). PCR products derived from seven patients with liver disease and three healthy controls were cloned and then subjected to phylogenetic analyses, which failed to link a virulent strain of TTV to severe liver disease. TTV infection was further assessed in an additional 148 subjects with normal liver biochemical tests, including 30 newborns (sera collected from the umbilical cord), 23 infants, 16 preschool children, 21 individuals of an age prior to that of sexual experience (aged 6 to 15 years), 15 young adults (aged under 30 years), and 43 individuals older than 30 years. The rates of TTV viremia were 0, 17, 25, 33, 47, and 54%, respectively. These findings suggest that TTV is transmitted mainly via nonparenteral daily contact and frequently occurs very early in life and that TTV infection does not have a significant effect on liver disease.

Case report: Clostridium septicum infection presenting as liver abscess in a case of choriocarcinoma with liver metastasis.

Clostridium septicum is an anaerobic, gram-positive bacillus. Infection with this organism has a known association with malignant diseases, especially colon and haematological cancers. Clostridium septicum is rarely found to be a pathogen of liver abscess. Herein, we report the case of a female choriocarcinoma patient with liver metastasis in which C. septicum infection presented as a gas-forming liver abscess. This case and previous reports indicate that once C. septicum is identified as a pathogen in liver abscess, metastatic liver tumours should be highly suspected.

Emergence of an S gene mutant during thymosin alpha1 therapy in a patient with chronic hepatitis B.

The presence of a hepatitis B virus S gene mutant was investigated in a patient being treated with thymosin alpha1. He was seropositive for hepatitis B e antigen throughout therapy but was intermittently seronegative for hepatitis B surface antigen (HBsAg) by an RIA. Sequence analysis revealed an S gene mutant in HBsAg-seronegative serum with two consecutive amino acid substitutions: threonine115-to-isoleucine and threonine116-to-asparagine, whereas no amino acid substitution or deletion was found in the pre-S region. A site-directed mutagenesis experiment confirmed that these mutations were responsible for the failure to detect HBsAg. In summary, an S gene mutant was identified in an HBsAg-seronegative patient. The mutations were located outside the putative "a" determinant. The emergence of an S gene mutant during thymosin alpha1 treatment suggests that enhanced host immunity against HBsAg may play a role in its antiviral activity.

Identification of a novel strain of hepatitis E virus responsible for sporadic acute hepatitis in Taiwan.

Hepatitis caused by the hepatitis E virus (HEV) is a self-limited disease and occurs most frequently as epidemic or sporadic hepatitis in developing countries. The role of HEV in sporadic acute hepatitis in areas without a history of hepatitis E epidemics is obscure. Recently, it was found that more than 10% of the patients with acute non-A, non-B, non-C hepatitis in Taiwan were associated with an acute HEV infection. Nucleotide sequences of the regions within the first open reading frame of HEV were determined in four cases and were 96.7-100% identical to each other. As compared to the isolates from China, Pakistan, Burma, India, Africa, and Mexico, the similarities were, however, only 71.7-79.3%. Phylogenetic analysis revealed that the four Taiwan isolates were categorized as a novel HEV group (the Taiwan strain), which was distinct from all of the strains isolated from other parts of the world. In addition, the isolates from China, Burma, India, and Pakistan were catalogued as the second genotype of HEV (the Asian strain), and the Mexican isolate as the third (the Mexican strain). The African isolate was more related to the Asian type and might be a subtype of the Asian strain. A simple genotyping method by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) is described. The findings also support the hypothesis that HEV may be responsible for some sporadic acute non-A, non-B, non-C hepatitis in other developed countries.

Change of thermal stability of colicin E7 triggered by acidic pH suggests the existence of unfolded intermediate during the membrane-translocation phase.

Purified colicin E7 was analyzed by CD spectrum and gel filtration chromatography in a mimicking membrane-translocation phase. It was found that the CD spectra of colicin E7 at pH 7 and pH 2.5 were similar. Although the melting temperature of the protein shifted from 54.5 degrees C to 34 degrees C at low pH, the thermal denaturation curves of colicin E7 at different pH conditions still fit a two-state model. These experimental results imply that a minor structural change, triggered by acidic pH, for instance, may reduce the energy required for protein melting. In contrast to the minor change in secondary structure at different pH conditions, we observed that, in vitro, all monomeric colicin E7s converted into multimer-like conformations after recovering from the partial unfolding process. This multimeric form of colicin can only be dissociated by formamide and guanidine hydrochloride, indicating that this protein complex is indeed formed by aggregation of the monomeric colicins. Most interestingly, the aggregated colicins still perform in vivo bacteriocidal activity. We suggest that in a partial unfolding state the colicin is prepared for binding to the specific targets for translocation through the membrane. However, in the absence of specific targets in vitro these unfold intermediates may therefore aggregate into the multimeric form of colicins.

Clinical and virological course of chronic hepatitis B virus infection with hepatitis C and D virus markers.

OBJECTIVE: Hepatitis B, C, and delta virus (HBV, HCV, HDV) share similar transmission routes; thus, dual or triple infections may occur and even persist in the same patient. However, little is known about the presentations and course of chronic HBV infection with HCV and HDV markers, which this study examined. METHODS: Antibodies against HCV (anti-HCV) and HDV (anti-HDV) were assayed as appropriate in patients with HBV infection. The clinical, pathological, and virological presentations as well as the course of the disease in patients with HBV/HDV/HCV triple infection markers were then reviewed. RESULTS: A total of 60 patients, 51 men and nine women, age 19-67 yr (mean 45.9+/-1.6 yr) were identified. Of these 60 patients, five (8.3%) were HBeAg positive and 10 (16.7%) cirrhotic at entry, 30 (50%) presented with acute superinfection (HCV or HDV, or both) and the remaining 30 presented with chronic liver disease. On presentation, 16 (53.3%) of the 30 patients with acute superinfection showed hepatic decompensation and eight (26.7%) died. In contrast, only one of the patients with "chronic liver disease" presented with hepatic decompensation. Of the 42 patients followed up for 1-15 (mean, 4.7+/-0.6) yr, 45.2% showed remission and 19% showed HBsAg seroclearance, whereas 12.5% of the 32 noncirrhotics developed cirrhosis and three of the nine cirrhotics became decompensated. At the end of follow-up, 29 patients (69.9%) were still seropositive for HCV-RNA but only nine (22.5%) were seropositive for HDV-RNA and five (12.5%) were seropositive for HBV-DNA. CONCLUSIONS: These results suggest that infection with HBV, HCV, and HDV triple markers is a severe disease in acute superinfection stage but that the course is relatively benign, slowly progressive, and usually dominated by HCV.

Primary hepatic angiosarcoma: report of a case involving environmental arsenic exposure.

Hepatic angiosarcoma is a rare malignant tumor with a rapidly fatal course. It has become a subject of interest because of its intimate relationship with environmental carcinogens, such as thorium dioxide (Thorotrast), vinyl chloride monomer, and arsenic. We describe a case of a chronic hepatitis B surface antigen carrier, with a 20-year history of environmental exposure to arsenical-containing agricultural herbicides and bactericides, who developed a hepatic angiosarcoma. He died due to rupture of the hepatic angiosarcoma with acute hemoperitoneum 9 weeks after initial diagnosis. This is a rare case of primary hepatic sarcoma, especially in Taiwan where hepatocellular carcinoma is endemic. This case not only serves to give more evidence of the relationship between hepatic angiosarcoma and arsenical exposure, but also demonstrates the key point in the differential diagnosis of liver tumors. Increased familiarity with this disease will facilitate correct diagnosis and help to improve management of the condition in the future.

High-performance liquid chromatographic determination of clindamycin in human plasma or serum: application to the bioequivalency study of clindamycin phosphate injections.

This paper presents an assay of clindamycin phosphate injection in human plasma or serum. A 0.5-ml volume of plasma was used with the internal standard, propranolol. The sample was loaded onto a silica extraction column. The column was washed with deionized water and then eluted with methanol. The eluates were evaporated under nitrogen gas. The residue was reconstituted with the mobile phase and injected onto the high-performance liquid chromatographic system: a 5-micron, 25 cm X 4.6 mm I.D. ODS2 column was used with acetonitrile, tetrahydrofuran and 0.05 M phosphate buffer as the mobile phase and with ultraviolet detection at 204 nm. A limit of quantitation of 0.05 microgram/ml was found, with a coefficient of variation of 11.6% (n = 6). The linear range is between 0.05 and 20.00 micrograms/ml and gives a coefficient of determination (r2) or 0.9992. The method has been successfully applied to the bioavailability study of two commercial preparations of clindamycin phosphate injection (300 mg each) in twelve healthy adult male volunteers.

Cloning and characterization of the extreme 5'-terminal sequences of the RNA genomes of GB virus C/hepatitis G virus.

The extreme 5'-terminal sequences of the GB virus C/hepatitis G virus (GBV-C/HGV), containing elements essential for regulation of viral gene expression and replication, have not been determined. By using a RNA-ligase-mediated RACE (rapid amplification of the cDNA ends) procedure, we have cloned the extreme 5'-terminal sequences of the viral genome from the serum of three Taiwanese patients. Sequence analysis of the 5' noncoding region in alignment with one West African and two American isolates showed that (i) a consensus 5'-end sequence was cloned; (ii) about 97% of sequences were homologous among the three Taiwan isolates and also between the two American isolates, whereas about 90% of sequences were homologous among the isolates from the three different geographic areas; (iii) the sequence heterogeneity related to geographic separation is confined mainly to three domains; and (iv) a potential hairpin structure, resembling the hairpin structure found in the 5' end of hepatitis C virus genome, was detected in the 5' end of the noncoding region. Our data support the hypotheses that (i) the extreme 5' end of the hepatitis GBV-C/HGV viral genome has been cloned, (ii) there are different genotypes correlated with geographic separation, and (iii) the viral translation and replication mechanisms may be similar to that of hepatitis C virus and pestiviruses. Our data have not only shed light on the viral replication mechanism but also offer information for selection of optimal primer sequences for the detection and genotyping of the hepatitis GBV-C/HGV virus by PCR assays.

A novel role of ImmE7 in the autoregulatory expression of the ColE7 operon and identification of possible RNase active sites in the crystal structure of dimeric ImmE7.

Site-specific cleavage of mRNA has been identified in vivo for the polycistronic colicin E7 operon (ColE7), which occurs between G and A nucleotides located at the Asp52 codon (GAT) of the immunity gene (ceiE7). In vitro, this specific cleavage occurs only in the presence of the ceiE7 gene product (ImmE7). The crystal structure of dimeric ImmE7 has been determined at 1.8 A resolution by X-ray crystallographic analysis. We found that several residues located at the interface of dimeric ImmE7 bear surprising resemblance to the active sites of some RNases. These results suggest that dimeric ImmE7 may possess a novel RNase activity that cleaves its own mRNA at a specific site and thus autoregulates translational expression of the downstream celE7 gene as well as degradation of the upstream ceaE7 mRNA.