TCF19 aggravates the malignant progression of colorectal cancer by negatively regulating WWC1.

OBJECTIVE: The aim of this study was to clarify the role of TCF19 in influencing the malignant progression of colorectal cancer (CRC) by negatively regulating WWC1. PATIENTS AND METHODS: Relative expression levels of TCF19 and WWC1 in CRC tissues and cells were detected by quantitative Real-Time Polymerase Chain Reaction (qRT-PCR). The prognosis of CRC patients was assessed by the Kaplan-Meier method. Meanwhile, the correlation between TCF19 and pathological indexes of CRC patients was evaluated. Regulatory effects of TCF19/WWC1 on viability, colony formation ability, and migration in HT29 and HCT-8 cells were evaluated. Finally, rescue experiments were conducted to elucidate a negative feedback loop of TCF19/WWC1 in influencing the progression of CRC. RESULTS: TCF19 was significantly up-regulated in CRC, while WWC1 was down-regulated. High-level TCF19 or low-level WWC1 indicated worse survival of CRC patients. Besides, TCF19 expression level was positively correlated with the occurrence of distant metastasis in CRC. Silence of TCF19 significantly attenuated proliferative and migratory capacities of HT29 cells. However, overexpression of TCF19 yielded the opposite trends in HCT-8 cells. WWC1 expression was negatively regulated by TCF19 in CRC tissues. In addition, knockdown of WWC1 abolished the regulatory effect of TCF19 on CRC cells. CONCLUSIONS: TCF19 is closely correlated with the occurrence of distant metastasis and poor prognosis of CRC. Furthermore, it aggravates the malignant progression of CRC via negatively regulating WWC1.

MK-571 attenuates kidney ischemia and reperfusion-induced airway hypersensitivity in rats.

OBJECTIVE: Reperfusion of the rat kidney has been shown to up-regulate cysteinyl leukotriene-1 receptor, an asthma-associated gene in human bronchioles, and increase expression of leukotriene D4. In this study, we aimed to investigate the efficacy of MK-571, a leukotriene D4 inhibitor, against hypersensitivity induced by kidney ischemia and reperfusion (I/R)-associated acute kidney injury. METHODS: Sprague-Dawley male rats were divided into 3 study groups: a sham-operated group, a kidney I/R group, and a group treated with MK-571 before the kidney I/R injury: MK-571 (5 mg/kg) was administered intraperitoneally 15 minutes before ischemia and every 12 hours after reperfusion up to 24 hours. Ischemia was conducted by bilateral occlusion of renal pedicles for 45 minutes, followed by releasing the clamps and closing the abdominal incision. Respiratory function was tested 24 hours after reperfusion, with the use of a 2-chamber whole body plethysmograph for conscious rats. Blood samples, pulmonary bronchoalveolar lavage fluid, and lung tissues were collected at the end of study. In 10 rats, urine was collected at baseline and the end of study. RESULTS: Compared with the sham group, kidney I/R injury markedly increased enhanced pause (Penh) index during methacholine challenge test (P < .05), suggesting airway hypersensitivity; it also increased in inflammatory response and levels of hydroxyl radical production and lipid peroxidation in the lungs. In contrast, in MK-571-treated rats, Penh was muted during methacholine challenge test (P < .05). CONCLUSIONS: Kidney I/R injury induces airway hypersensitivity to methacholine challenge test and inflammatory response and oxidative stress in the lungs. Treatment with MK-571, a leukotriene D4 inhibitor, effectively attenuates airway hypersensitivity, pulmonary inflammatory response, and lung and kidney injury.

Characterization of a 120-Kilodalton pre-S-binding protein as a candidate duck hepatitis B virus receptor.

Infection by human and animal hepadnaviruses displays remarkable host and tissue tropism. The infection cycle probably initiates with binding of the pre-S domain of viral envelope protein to surface receptors present on the hepatocyte. Three types of neutralizing monoclonal antibodies against duck hepatitis B virus (DHBV) have their binding sites clustered within residues 83 to 107 of the pre-S protein, suggesting that this region may constitute a major receptor binding site. A 170- or 180-kDa duck protein (p170 or gp180) which binds DHBV particles through this part of the pre-S sequence has been identified recently. Although the p170 binding protein is host (duck) specific, its distribution is not restricted to DHBV-infectible tissues. Using the pre-S protein fused to glutathione S-transferase and immobilized on Sepharose beads, we have now identified an additional binding protein with a size of 120 kDa (p120). p120 expression is restricted to the liver, kidney, and pancreas, the three major organs of DHBV replication. While optimal p170 binding requires an intact pre-S protein, binding to p120 occurs much more efficiently with a few N- or C-terminally truncated forms. The p120 binding site was mapped to residues 98 to 102 of the pre-S region, which overlaps with a cluster of known virus-neutralizing epitopes. Site-directed mutagenesis revealed residues 100 to 102 (Phe-Arg-Arg) as the critical p120 contact site; nonconservative substitution in any of the three positions abolished p120 binding. Double mutations at positions 100 to 102 markedly reduced DHBV infectivity in cell culture. Short pre-S peptides covering the clustered neutralizing epitopes (also p170 and p120 binding sites) reduced DHBV infectivity in primary duck hepatocyte cultures. Thus, p120 represents a candidate component of the DHBV receptor complex.

Single-step nested polymerase chain reaction for detection of different genotypes of hepatitis C virus.

Hepatitis C virus (HCV) exhibits considerable sequence variability and circulates in the blood at extremely low levels. Current methods for detecting HCV RNA are based mostly on nested polymerase chain reaction (PCR), in which part of the first amplification product is reamplified in the second tube by an internal primer pair. A novel nested PCR method was developed in which the two successive amplification processes are carried out in the same tube with a single step of physical manipulation. Careful selection of highly conserved sequences of the 5' noncoding region as primers enabled successful detection of all three major genotypes circulating in France, including the one with variation in this region. Retaining high sensitivity of the conventional nested PCR, the novel method reduced greatly the risk of carry-over contaminations. It was also cost- and time-saving. The one-step nested PCR method is especially suitable for routine diagnosis of HCV infection in clinical laboratories.

Characterization of the third most common genotype of hepatitis C virus in France.

We have previously identified the two major hepatitis C virus (HCV) genotypes prevalent in France (type I and type II). We report here the identification and partial characterization of a new HCV genotype with a highly divergent 5' noncoding (NC) region and a structural protein region. This genotype showed only 93-94% sequence identity with either type I or type II HCV in the 5' NC region. Sequence analysis of the structural protein region revealed extremely low sequence homology with all the four major HCV genotypes: 86-89% for the core protein and 56-69% for the envelope protein. However, further analysis revealed that this new genotype was very similar to the genotype 3a described most recently. Screening of 150 clinical samples with genotype-specific oligoprobes revealed prevalence of this genotype in 12% of the French samples with a significant association with drug addiction and a good response to interferon therapy. These results may have implications for the diagnosis of HCV infection and the design of HCV vaccines.

Hepatitis C virus genotypes in France: comparison of clinical features of patients infected with HCV type I and type II.

Two major hepatitis C virus genotypes, F1 and F2, corresponding to hepatitis C virus type I and type II respectively, were found in France. To investigate the correlation between infection with these genotypes (F1 and F2) and clinical features of patients, serum samples proven to be hepatitis C virus positive by polymerase chain reaction amplification on 5' non-coding region were further amplified in the NS3 region with nested polymerase chain reaction. The NS3-polymerase chain reaction products were Southern blotted and hybridized with specific probes to identify the genotype of hepatitis C virus. Of 70 samples 64 were NS3-polymerase chain reaction positive. Twenty-eight (40%) samples were hepatitis C virus type I (F1) and 34 (49%) were hepatitis C virus type II (F2), while one sample (HB) hybridized with both probes and another (HN) hybridized with neither. Some samples were sequenced, with results consistent with those of hybridization. The HB sample was related more to hepatitis C virus type II than to type I and the HN sample was divergent from both type I and type II genotypes. Clinical profiles of patients infected with hepatitis C virus type I and type II were compared. Type I infected patients were younger (p < 0.01) and more often male (p < 0.05) than those of the type II group. Nine of 28 patients in the type I infected group had a history of drug abuse, whereas none did in the type II group. Five of 22 (23%) type I infected patients and 19 of 32 (59%) type II infected patients had cirrhosis (p < 0.01).(ABSTRACT TRUNCATED AT 250 WORDS)

Heterogeneity of hepatitis C virus genotypes in France.

The genotypes of French hepatitis C virus (HCV) isolates were investigated by amplification of a domain from the non-structural region 3 (NS3) using nested PCR, followed by hybridization with two genotype-specific probes, F1 (HCV type I-specific) and F2 (HCV type II-specific). Among 119 HCV RNA-positive sera, 91% of samples were NS3 PCR positive. Most samples (83.2%) hybridized with one or the other probe only, whereas a few samples (4.2%) hybridized with both F1 and F2 probes (HB). A small percentage (3.4%) of samples appeared unable to hybridize with either probe (HN). For some of these samples (HB1, HB2, HN1, HN2, HN3, HN4), part of the NS3, core and envelope regions were sequenced and the corresponding deduced consensus sequences were compared with those of prototype isolates of the four HCV genotypes (types I to IV). A phylogenetic tree was constructed to illustrate the relationship between these isolates. The results obtained showed that (i) HN4 appears to be more closely related to type III than to type IV HCV genotypes, which suggests that in France there may exist additional although minor genotypes besides the two major types, F1 and F2. (ii) HB1, HB2, HN1, HN2 and probably HN3 belong to the type II HCV genotype. The association between sequence diversity and putative biological difference for isolates within the same genotype remains to be elucidated.

Identification of the third major genotype of hepatitis C virus in France.

We have previously found type I and type II hepatitis C virus (HCV) as the predominant HCV genotypes in France. Here we report on the identification and partial characterization of a third major genotype there. This genotype showed only 93-94% sequence conservation to either type I or type II HCV in the 5' noncoding region. The point mutations changed the small open reading frames but not the proposed secondary structure of this region. Sequence of the structural protein region also differed significantly from other genotypes. The new genotype was found in 12% of HCV infections in France. Rapid method for the identification of this genotype was developed.

Hepatitis B virus genotype A rarely circulates as an HBe-minus mutant: possible contribution of a single nucleotide in the precore region.

The emergence of HBe-minus hepatitis B virus (HBV) mutants, usually through a UAG nonsense mutation at codon 28 of the precore region, helps the virus to survive the anti-HBe immune response of the host. Host and viral factors that predispose to the emergence of such mutants are not well characterized. The fact that the precore region forms a hairpin structure essential for the packaging of viral pregenomic RNA may explain the extremely high prevalence of the UAG mutation at codon 28. It converts a wobble U-G pair in the packaging signal between nucleotide 3 of codon 15 (CCU) and nucleotide 2 of codon 28 (UGG) into a U-A pair. Since genotype A of HBV has a CCC sequence at codon 15, the UAG mutation would, instead, disrupt a C-G pair present in the wild-type virus. This alteration was shown by transfection experiments to greatly compromise the packaging of pregenomic RNA. The implication of this finding was elucidated by molecular epidemiological studies. Genotype A was found to be the most prevalent genotype in the wild-type virus populations in France but was found in only 1 of the 46 isolates of HBe-minus mutants found there. These mutants were contributed chiefly by genotype D, the second most prevalent genotype in France, which is characterized by a CCU sequence at codon 15. The role of the single nucleotide at codon 15 was confirmed by the finding of the single genotype A isolate in which both wild-type and mutant viruses were present. Interestingly, nearly all of the mutants had a codon 15 sequence of CCU instead of the CCC present in the wild-type viruses. Our results suggest that genotype A of HBV rarely circulates as HBe-minus mutants, probably because of a requirement for a simultaneous sequence change at codon 15. These data, together with the virtual absence of genotype A in the Chinese samples examined, may provide some insights into the uneven prevalence of HBe-minus mutants in the world.

Evidence for a base-paired region of hepatitis B virus pregenome encapsidation signal which influences the patterns of precore mutations abolishing HBe protein expression.

In two natural HBe-minus hepatitis B virus mutants, expression of HBe protein was abrogated by a nonsense mutation at precore codon 28 and a frameshift mutation at codon 29, respectively. Both mutants contained an additional nucleotide substitution(s) which was found by transfection experiments to be required for efficient packaging of pregenomic RNA. The observed mutational profiles were consistent with the presence of a base-paired region of the pregenome encapsidation signal overlapping the HBe-coding sequence. Results obtained with artificial mutants with significant changes in the primary sequence suggested that base pairing is required but insufficient for efficient pregenome packaging. However, the predicted first four base pairs of the stem are dispensable.

Replication capacities of natural and artificial precore stop codon mutants of hepatitis B virus: relevance of pregenome encapsidation signal.

The emergence of hepatitis B virus variants unable to express HBe protein during late stage of viral infection may represent an important mechanism of viral persistence. The molecular mechanisms responsible for the elimination of HBe expression are nonsense or frameshift mutations or initiation codon mutations in part of its coding sequence, the precore region. So far only 2 of the 29 precore amino acid codons have been found mutated to stop codons in nature, although a total of 10 codons are convertible to stop codons by single nucleotide changes. Since the HBe-coding sequence is largely overlapped by the pregenome encapsidation signal (epsilon signal), a recently found cis-acting element required for the packaging of pregenomic RNA, the absence of other potential nonsense mutants could result from their impairment of the epsilon signal. Seven such potential stop codon mutants were constructed and tested for replication capacities by transfection into a hepatoma cell line. Five mutants were replication competent, but at levels lower than that of a prevalent natural stop codon mutant. The remaining two mutants were completely defective in DNA replication, which clearly explained why these two mutants are not found in nature. Northern blot analysis revealed wild-type levels of RNA transcription by these two mutants but complete lack of packaged pregenomic RNA. Additional studies lent further support to the importance of the epsilon signal in pregenome encapsidation and suggested relaxed sequence requirements for the computer-predicted hexanucleotide bulge region as compared to the hexanucleotide loop of the signal.

Lack of pre-C region mutation in woodchuck hepatitis virus from seroconverted woodchucks.

Woodchuck hepatitis virus, which shares a large degree of homology with human HBV, was examined for indications of mutational variants. No alteration in the pre-C region was found, but as in HBV, viral DNA could still be detected by PCR after seroconversion to anti-WHe.

Generation of a 1.5-kb cDNA fragment of the hepatitis C virus genome by overlap extension.

Hepatitis C virus (HCV) is a recently identified RNA virus responsible for most of non-A, non-B hepatitis. Genetic analysis of HCV sequences and their encoded proteins has been hampered by the difficulty in obtaining cDNA fragments of sufficient lengths: construction of cDNA library requires technical expertise while amplification by the polymerase chain reaction (PCR) usually yields fragments of less than 400 base pairs. In this report we have generated a 1.5-kb HCV cDNA fragment by overlap extension of smaller PCR fragments and by ligation through restriction sites. Sequencing of the cloned fragment confirmed the absence of significant sequence alteration produced by this procedure. Overlap extension may represent an easy method for generating relatively large HCV cDNA clones for the expression of HCV-encoded proteins.

Two French genotypes of hepatitis C virus: homology of the predominant genotype with the prototype American strain.

A hepatitis C virus (HCV) cDNA covering part of the nonstructural region, NS3, was amplified from the serum of 50 out of 76 French non-A, non-B hepatitis patients by the nested polymerase chain reaction (PCR). Determination of a 407-bp sequence from four such cases revealed the presence of two different virus genotypes, F1 and F2, which exhibited 19-20% sequence divergence. F1 was represented by three of the four isolates and showed a sequence homology of about 97.5% to the prototype American HCV isolate, but of only 79% to a reported Japanese isolate. In contrast, F2 had 91.6% homology to the Japanese isolate, but only 81% homology to the prototype American HCV. PCR products from the 50 samples were hybridized with labeled F1 and F2 fragments under stringent conditions; results indicated the F1-related strain(s) as the major HCV genotype. Furthermore, a total of 1477 bp of sequence has been determined for one of the isolates belonging to the F1 category. These results will have implications for the PCR detection of HCV infection and production of HCV vaccines, especially for European countries.

In vitro replication competence of a cloned hepatitis B virus variant with a nonsense mutation in the distal pre-C region.

Hepatitis B virus (HBV) variants with a nonsense mutation in the distal pre-C region have been detected in patients positive for anti-HBe, and the complete nucleotide sequence of one cloned pre-C variant has been determined. Transfection of this HBV variant clone into the human hepatoma cell line HepG2 resulted in the appearance of major HBV transcripts, replicative forms of viral DNA evidenced by both molecular hybridization and endogenous DNA polymerase assay, as well as the expression and secretion of HBsAg and HBcAg particles. Western blotting revealed only the 21-kDa HBcAg but not the 17-kDa HBeAg. These results demonstrate the replication capacity of the HBV variant with a nonfunctional pre-C region despite its inability to express HBeAg.

Rapid screening for bacterial colonies harbouring tandem hepatitis B virus sequences by an oligonucleotide probe.

Transfection of the hepatitis B virus (HBV) genome requires the cloning of tandem HBV sequences into a plasmid vector, which is usually screened for by restriction enzyme digestion of plasmid minipreparations from at least a dozen bacterial colonies. We describe a simple alternative screening method based on in situ hybridization of bacterial colonies with a [32P]-labelled synthetic oligonucleotide which spans the head-to-tail junction site of two tandem HBV molecules. The accurate detection by the oligoprobe is confirmed by enzymatic digestion.

Evidence of two major genotypes of hepatitis C virus in France and close relatedness of the predominant one with the prototype virus.

Hepatitis C virus (HCV) cDNA sequence in the nonstructural region NS3 was amplified from the serum of 66% French non-A, non-B hepatitis patients by the nested polymerase chain reaction. A 407 base-pair sequence was determined from four such cases, which revealed the presence of two different virus genotypes F1 (three cases) and F2 (one case) with 19-20% sequence divergence. F1 showed close homology (97.5%) to the prototype US isolate, but only limited (79%) homology to the reported Japanese isolates. In contrast, F2 had 91.6% homology to the Japanese isolate, but only 81% homology to the prototype US virus. Hybridization of the amplified products from 50 French samples with labeled F1 and F2 fragments suggested the F1-related strain(s) as the major hepatitis C virus genotype. Further studies involving a greater variety of samples will confirm whether the F1-related strain is the predominant hepatitis C virus strain circulating in France. Such data will have important implications for the PCR detection of HCV infection and production of HCV vaccines.

In vitro and in vivo replication capacity of the precore region defective hepatitis B virus variants.

Although the precore region defective hepatitis B virus variants have been implicated in chronic liver disease and fulminant hepatitis, our knowledge on the molecular biology of these variants is still limited. Using an in vitro transfection assay, we confirmed the replication competent but HBeAg-negative nature of the major variants containing a TAG stop codon in the distal precore region associated with one or two point mutations. Transfection of the two-point-mutated variant into a chimpanzee induced serological responses including anti-HBc and anti-HBs. Interestingly, anti-HBe response was found in the absence of HBeAg antigenemia, suggesting that anti-HBe can be stimulated by degraded HBc. Using the rabbit reticulocyte system the possible effect of the different precore region mutations on the expression of HBcAg from precore- and core-mRNAs was also studied.

Active hepatitis B virus replication in the presence of anti-HBe is associated with viral variants containing an inactive pre-C region.

Although rise of anti-HBe immunity in the course of hepatitis B virus (HBV) infection is generally followed by clearance of the infectious virions, ongoing chronic liver disease with circulating virions has been repeatedly observed in a significant number of anti-HBe patients, especially in Mediterranean countries. To investigate the possible role of HBV variants, we cloned HBV DNA from the serum of three such anti-HBe cases. Comparative restriction mapping of HBV clones suggested circulation of different HBV genomes in the three cases. DNA sequencing revealed an inactive pre-C region in all 11 HBV clones derived from the three cases, either as one or two point mutations in the 3' terminus generating an in-frame TAG stop codon, or a 1 nucleotide insertion in the 5' terminus resulting in frameshift mutation. Furthermore, for one clone the complete 3182 nucleotide sequence was determined and no significant mutation was found in the remainder of the genome. We conclude that chronic hepatitis cases positive for anti-HBe are associated with HBV variants containing an inactive pre-C region and hence cannot synthesize pre-C region-derived HBeAg. This finding may provide a molecular explanation for the continued viral replication despite presence of anti-HBe immunity.