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.

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.

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.

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.

Regulation of polyadenylation of hepatitis delta virus antigenomic RNA.

Hepatitis delta virus (HDV) is a subviral agent with a small RNA genome that is replicated in the nucleus of an infected cell. During genome replication, there is the synthesis of a complementary RNA, known as the antigenome, and also of a smaller complementary species that is polyadenylated and acts in the cytoplasm as the mRNA for the only known HDV protein, the delta antigen. We have carried out an examination of the cis- and trans-acting elements that regulate the polyadenylation process involved in the synthesis of this mRNA for the delta antigen. Our experimental approach has been to study the processing of nascent antigenomic RNA as it occurs in transfected cells via DNA-directed RNA synthesis, in the absence of genome replication. Three conclusions have been made. (i) The polyadenylation process occurs independent of the functionality of a unique self-cleavage domain located just 3' of the polyadenylation site. (ii) RNA transcripts that proceed beyond the polyadenylation site can be stabilized by the self-cleavage reaction. Thus, a single transcription initiation event can lead not only to the mRNA species but also to at least one more stable RNA species. (iii) If the nascent RNA species can fold on itself, into the so-called rodlike structure, then the presence of the delta antigen leads to a major suppression of polyadenylation. These results are incorporated into a more detailed model of the replication of the HDV genome.

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.

The antigen of hepatitis delta virus: examination of in vitro RNA-binding specificity.

The only known protein of hepatitis delta virus (HDV), the delta antigen, is found both within virus particles and within the nucleus of the infected cell, where it has one or more roles essential for RNA genome replication. Others have demonstrated that the antigen has the ability, in vitro, to specifically bind HDV RNA species. We report a further examination of this phenomenon, using partially purified recombinant protein, expressed as a fusion with the staphylococcal protein A. From Northwestern (RNA-immunoblot) analyses with both complete and various subdomains of HDV genomic and antigenomic RNAs, we found that a necessary feature for specific binding was that the RNA be able to fold to some extent into the so-called rodlike structure; this structure is a predicted intramolecular partial base-pairing of the circular RNA, with about 70% of all bases involved, so as to produce an unbranched rodlike structure. Six different subregions of the HDV rodlike structure, three on the genomic RNA and three on its complement, the antigenomic RNA, were tested and found to be sufficient for antigen binding. However, features in addition to the rodlike structure may also be necessary for specific binding, because we found that a similar structure present in the RNA of the potato spindle tuber viroid did not allow binding.

Mapping of transcriptional start sites of the cea and cei genes of the ColE7 operon.

Two transcriptional start sites were identified 77 and 78 nucleotides upstream of the translation initiation codon of the colicin E7 gene (ceaE7). The guanosine nucleotide located at the fifth position of the SOS box is probably a universal transcriptional start site of all E group colicins. Major and minor transcripts of the immunity gene (cei) are initiated at the 3' end of the cea gene. Relative to the -10 sequence, CAAAAT, of the major ceiE7 promoter, the corresponding region of the cei gene of other E group colicins has an increased content of guanosine nucleotides. However the -10 sequence of the minor ceiE7 promoter, TATGAT, was found to be conserved in other colicin promoters. The results indicate that the structure of the major promoter of the ceiE7 gene is unique among the E group colicins.

Hepatitis delta virus genome replication: a polyadenylated mRNA for delta antigen.

Hepatitis delta virus (HDV) replicates its genome in the nucleus of an infected cell. However, an unsolved problem has been the identification in the cytoplasm of a putative mRNA for the synthesis of the only virus-coded protein, the delta antigen. We now report the characterization of an 800-base RNA that is cytoplasmic, polyadenylated, and antigenomic and that should direct the translation of the delta antigen. This RNA was about 500 times less abundant than full-length genomic RNA. We mapped the predominant 5' terminus and also the 3' site at which the poly(A) is added. At a point 15 to 20 bases upstream of the poly(A) addition site is the sequence AAUAAA, which could have been used as a signal for the polyadenylation. When an infectious cDNA clone of the whole HDV genome was changed at this site to UUUAAA, the clone was no longer infectious and it was unable to direct the synthesis of the delta antigen. These findings provided additional evidence that the polyadenylated RNA was at least the predominant method for the expression of the delta antigen. Apparently the HDV RNA was processed as if it were a host mRNA polymerase II transcript, although this did not necessarily indicate that HDV RNA was transcribed with this enzyme.

Role of two forms of hepatitis delta virus antigen: evidence for a mechanism of self-limiting genome replication.

The replication of the RNA genome of hepatitis delta virus is greatly facilitated by the presence of the only known virus-coded protein, the delta antigen. Most, if not all, infections are characterized by the presence of two electrophoretic forms of the delta antigen. These forms correspond to polypeptide lengths of 195 and 214 amino acids which are encoded by genomes with different nucleotide sequences. We used cDNA transfections to investigate the functions of these two forms of the delta antigen. We found that only the small form of delta antigen supported hepatitis delta virus genome replication and that the large form acted as a dominant negative repressor of such replication. This inhibition was potent. For example, the amount of genome replication was reduced eightfold when as little as 10% of the delta antigen was present as the large form. One interpretation of our results is that the delta antigen normally functions as part of a multimeric structure. In addition, our data suggest that synthesis of the large form, either during genome replication in cultured cells or even during infection in animals, may suppress delta replication, possibly leading to a self-limiting infection.

The roles of the delta antigen in the structure and replication of hepatitis delta virus.

The delta antigen is the only known protein encoded by hepatitis delta virus (HDV). The predicted protein is 195 amino acids in length, but for reasons that are not yet clear, there occurs during the replication of the HDV genome, a specific base change in the termination codon of this open reading frame. This leads to the synthesis of a form of the delta antigen that is 19 amino acids longer, with a total length of 214 amino acids. Studies are described which relate to the roles of these two forms of the delta antigen in genome replication and subsequent particle assembly.

Role of the ribozyme of hepatitis delta virus on the transcription after polyadenylation.

We have previously demonstrated that the ribozyme located 34 nucleotides downstream of the polyadenylation site on the antigenomic RNA of the hepatitis delta virus can stabilize the downstream transcript after polyadenylation. Here, we have reports on further investigations of the molecular mechanism of this stabilization effect and the potential role of the small and large delta antigens. We found that the downstream transcripts after polyadenylation were stabilized by the ribozyme independently of either the small or large delta antigen. The stabilization effect was abolished as the ribozyme activity was eliminated by mutations on either the enzyme domain or target site of the ribozyme. These findings suggested that it was the ribozyme activity rather than the RNA structure or the delta antigens that contributed to the stabilization effect.

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.

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 specific base transition occurs on replicating hepatitis delta virus RNA.

Three independent lines of evidence showed that when an infectious clone of hepatitis delta virus of known sequence was used to initiate genome replication, up to 41% of the genomes were specifically mutated in the amber termination codon (UAG to UGG) for the open reading frame of the delta antigen, thereby increasing the length of the predicted protein from 195 to 214 amino acids. This change was detected only on molecules that participated in RNA-directed RNA synthesis.

Serum levels of interferon-alpha and -gamma in acute and chronic hepatitis B virus infection.

To evaluate the potential implication of in vivo interferon production in the pathogenesis of different forms of acute and chronic hepatitis B virus infection, serum levels of interferon-alpha and -gamma were measured using immunoassay techniques in 20 patients with acute hepatitis B who subsequently cleared the virus (group Ia), 8 patients with acute hepatitis B who became HBsAg carriers (group Ib), 55 patients with chronic hepatitis B (group II), and 15 healthy controls. None of the controls had interferon-alpha or -gamma detectable in serum, while 15% and 100% of group Ia patients, 25% and 100% of group Ib patients, and 22% and 15% of group II patients, had raised serum levels of interferon-alpha and -gamma, respectively. Serum interferon-gamma was detected significantly more frequently in group Ia and Ib patients than in controls and in group II patients. Among patients with acute hepatitis B, serum levels of interferon-alpha and -gamma showed no significant difference between group Ia and group Ib patients. Among patients with chronic hepatitis B, interferon-alpha was detected significantly more frequently in patients with serum HBV-DNA (31.4% or 11/35) than in those without (5% or 1/20), whereas interferon-gamma was detected significantly more frequently in patients with chronic active hepatitis (28% or 7/25) than in those with chronic persistent hepatitis (3.3% or 1/30). In conclusion, in acute hepatitis B, serum levels of interferon-alpha and -gamma did not show a significant difference between patients who subsequently cleared the virus and those who became HBsAg carriers.(ABSTRACT TRUNCATED AT 250 WORDS)

Polyadenylation of the mRNA of hepatitis delta virus is dependent on the structure of the nascent RNA and regulated by the small or large delta antigen.

During the hepatitis delta virus (HDV) RNA replication, synthesis of either the mRNA for the delta antigen (HDAg) or the full-length antigenomic RNA is determined by selective usage of the potent poly(A) signal on the antigenome. To elucidate the regulatory mechanism, HDV cDNA cotransfection system was used to examine the potential effect of the secondary structure of the nascent RNA and that of the HDAg on HDV polyadenylation in transfected cells. We found that when the nascent RNA species could fold itself to form the rodlike structure, the HDV polyadenylation was suppressed 3 to 5 fold by the HDAg. In addition, we observed that the small and the large HDAg exerted a similar suppressive effect on the HDV polyadenylation, though they played different roles in HDV replication. We concluded that the HDV polyadenylation could be regulated by the structure of the nascent antigenomic RNA and by either the small or large HDAg.

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.

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.