[Hepatitis C virus genotyping by restriction fragment length polymorphism of polymerase chain reaction products generated with a HCV detection kit].

Highly conserved sequence in the 5' untranslated region(UTR) of hepatitis C virus(HCV) genome have been targeted by most nucleic acid amplification-based detection assays, such as Amplicor HCV test, a commercially available assay kit. In this study, we classified HCV genotypes by direct sequencing determination for 5' UTR of nested-PCR after Amplicor HCV test. Then, based on the results of sequence, RFLP analysis after digestion of the nested PCR fragments with Hae III or Sau 3AI to classify HCV genotype was evaluated. RFLP analysis distinguished the type 1, 2a and 2b. Only one of 29 samples was not classified by RFLP analysis due to the point mutation of Hae III recognition site. HCV genotypes commonly found in JAPAN were classified into three types, 1b, 2a, and 2b. Also, RFLP analysis requires fewer resources than serotype grouping test. Hence, the present method provides an adaptable and rapid HCV genotyping in clinical laboratory in JAPAN.

[Hepatitis B virus gene mutations in the sera of three patients with coexisting hepatitis B surface antigen and anti-surface antibody].

We analyzed gene mutations in the Hepatitis B virus of three virus carriers with coexisting Hepatitis B surface(HBs) antigen and anti-HBs antibody. Viral DNAs were extracted from sera and the pre-S, S and X(including core promoter and pre-core region) regions were amplified by PCR, and sequenced. Case 1 and Case 2 were positive for HBe antigen, while Case 3 was negative. All three cases were positive for HBe antibody and HBV DNA. In the S gene region, various point mutations were detected in all three cases. Mutations were clustered in the first hydrophilic loop region(codon 47-46) essential for the secretion of surface antigen. A few mutations were detected in 'a' loop(codon 124-147) of the S gene. None of the cases had an amino acid substitution of codon 145 of the S gene that is reported to be responsible for weak recognition by the HBs antibody. These data suggest the existence of hyper-variable sequence in S region, or otherwise result of low-fidelity of Taq DNA polymerase-reaction. Case 1 possessed a point mutation, T to C at nucleotide position 1753, in the region overlapping the coding region of the X gene and the CCAAT/enhancer binding protein(C/EBP) binding region within the core promoter region. Case 2 possessed both a large deletion(129 bp) in the pre-S1 and in-frame deletions of 15 and 27 bp in the pre-S2 region. Case 3 had an in-frame deletion of 30 bp in the pre-S2 region, and a point mutation in precore region. The point mutation, G to A at a nucleotide position 1986, converts Trp(TGG) to a stop codon TAG, and may contribute the fulminant hepatitis. These results suggest that the mutations in the pre-S, the core promoter, or the X gene may imply coexistence of the HBs antigen and antibody after seroconversion, while the point mutations in the S region are not likely to be responsible for the HBV escape mutant.

Enhanced expression of mRNA coding for the adrenaline-synthesizing enzyme phenylethanolamine-N-methyl transferase in adrenaline-secreting pheochromocytomas.

PURPOSE: In some pheochromocytomas, the tumors contain and secrete greater amounts of adrenaline than do normal adrenal medullas. It is not yet known how adrenaline synthesis is enhanced in the adrenaline-secreting pheochromocytomas. MATERIALS AND METHODS: As a first step toward understanding the molecular mechanisms by which adrenaline synthesis is controlled in these tumors, we measured the level of mRNA coding for the adrenaline-synthesizing enzyme phenylethanolamine N-methyl transferase (PNMT) and the content of adrenaline in the pheochromocytomas (n = 9), including 3 cases of the adrenaline-secreting type (one of the patients had bilateral pheochromocytomas), and in normal adrenal medullas (n = 7). We then measured the concentration of cortisol, which is thought to regulate the PNMT activity. Finally, we examined the expression of the mRNA for Egr-1, which was recently reported to be a transcriptional factor regulating PNMT gene expression. RESULTS: In the 4 tissue specimens from 3 adrenaline-secreting pheochromocytomas, the contents of adrenaline and the PNMT mRNA expression were considerably greater than those of the normal adrenal medullas. PNMT immunoreactivity was only detected in the adrenaline-secreting tumors. Three of the 4 specimens showed high concentrations of cortisol. To show the capacity for cortisol production locally in the pheochromocytoma tissues, we showed the expression of a glucocorticoid biosynthetic enzyme, 17alpha-hydroxylase, in the tumors by Western blotting. PNMT expression was found to be associated with 17alpha-hydroxylase expression in the tumors. The glucocorticoid receptor expression was also correlated with PNMT expression in the tumors and the expression of Egr-1 was also high in 3 of the 4 specimens. CONCLUSIONS: These findings indicate that adrenaline production in adrenaline-secreting pheochromocytomas is primarily controlled by the level of PNMT gene expression, and that the gene expression may be enhanced by both cortisol and Egr-1.

Transfusion-transmitted virus infection in China: prevalence in blood donors and in patients with liver diseases.

BACKGROUND: Prevalence of transfusion-transmitted virus (TTV) infection among blood donors and in patients with liver diseases in China was studied. METHODS: DNA was extracted from serum and amplified by seminested polymerase chain reaction with reported primer sets from a conserved region of the TTV genome. RESULTS: TT Virus DNA was detected in 55 of 196 blood donors (28%); 31% (40 of 127) in the north and 22% (15 of 69) in the south. TT Virus DNA was also detected in 14 of 31 patients (45%) with non-A-non-G fulminant hepatitis and in eight of 25 patients (32%) with non-A-non-G chronic hepatitis. The rate of TTV viraemia in these patients with liver disease was comparable to that in blood donors. TT Virus DNA sequencing of 12 isolates showed that the prevalence of genotype 2 was significantly higher than that reported in Japan (66.7 vs 2.6%, P < 0.001). Furthermore, genotyping assays based on restriction fragment length polymorphism were carried out on all 88 TTV DNA-positive samples. It was found that 42 isolates (47.7%) belonged to genotype 1 and 40 (45.5%) to genotype 2. It was of particular interest that the prevalence of genotype 1 in patients with non-A-non-G fulminant hepatitis was significantly higher than that in blood donors (10/14 vs 22/55, P < 0.05). CONCLUSIONS: The data indicate that TTV infection is common in China and that the pathogenic potential of TTV toward the liver (if any) may differ between genotypes.

Successive occupancy by immediate early transcriptional factors of the tyrosine hydroxylase gene TRE and CRE sites in PACAP-stimulated PC12 pheochromocytoma cells.

We previously demonstrated that pituitary adenylate cyclase-activating polypeptide (PACAP) coordinately upregulates the expression of the tyrosine hydroxylase (TH) and dopamine beta-hydroxylase (DBH) genes by activating the cyclic AMP (cAMP) and protein kinase C (PKC) signaling pathways. In this study, we examined the effects of PACAP on the expression of fos and jun immediate early gene (IEG) families, expression of which can be up-regulated by both PKC and cAMP signaling pathways, in rat pheochromocytoma cell line PC12 cells. PACAP potently stimulated the expression of c-fos, fosB junB and junD, but not c-jun mRNAs, at doses of 0.1-10 nM, as revealed by Northern blot analysis. The effects of PACAP on the expression of these mRNAs in PC12 cells was rapid (30-60 min) and dose-dependent. PACAP administration induced maximum expression of c-fos, fosB and junB mRNA after 60 min, and of junD mRNA after 8 h. Gel mobility shift assays using synthetic DNA oligonucleotides corresponding to the TH 5'-flanking region and nuclear extracts from PC12 cells demonstrated that PACAP enhanced formation of the specific protein complexes which bind to the TPA-responsive element (TRE) and cAMP-responsive element (CRE), respectively. Gel shift and supershift analyses showed that the TRE-binding factors and CRE-binding factors comprised fosB, c-fos, junB, and junD, and CRE-binding protein (CREB) and junD, respectively. JunB was dominant in the TRE-binding complexes at 4 h after addition of PACAP, whereas both JunD and JunB were dominant at 12 h. These results suggest that agonist occupancy of PACAP receptors activates transcriptional factors (Fos/Jun families and CREB) that interact with the TRE and CRE sites of the TH 5'-flanking region, contributing to transcriptional activation of TH gene.

Expression of mRNA coding for four catecholamine-synthesizing enzymes in human adrenal pheochromocytomas.

OBJECTIVE: To understand the molecular mechanisms by which catecholamine synthesis is controlled in pheochromocytomas--tumors that synthesize and release catecholamines, which are related to various clinical manifestations of the condition. METHODS: We measured the concentrations of mRNA coding for the catecholamine-synthesizing enzymes tyrosine hydroxylase, aromatic L-amino acid decarboxylase (AADC), dopamine beta-hydroxylase (DBH) and phenylethanolamine N-methyl transferase (PNMT) and for the catecholamine contents in 12 pheochromocytomas and 12 normal adrenal medullas. RESULTS: The mean content of total catecholamine and the beta-actin mRNA expression in the pheochromocytomas were almost the same as those in the normal adrenal medullas. However, the tyrosine hydroxylase, AADC and DBH mRNA concentrations in the pheochromocytomas were greater than those of the normal adrenal medullas. Conversely, the PNMT mRNA concentration in the pheochromocytomas was lower than that in the normal adrenal medullas. These differences are responsible for the difference in the proportions of catecholamines between pheochromocytomas and normal adrenal medullas. The constitutive expression of the catecholamine-synthesizing enzyme mRNAs varied in magnitude among the pheochromocytomas, and the tyrosine hydroxylase mRNA expressions correlated with the contents of total catecholamine in the tumors (r=0.964, P<0.0001). CONCLUSIONS: These findings indicate that catecholamine production in pheochromocytomas is primarily controlled by the level of gene expression.

[Comparison of three methods for quantitative measurement of hepatitis C virus].

Three assays for measuring hepatitis C virus(HCV) were compared with regard to sensitivity and correlations. The methods included were the Roche Amplicor HCV Monitor test(PCR), the branched DNA signal amplification assay(b-probe), and the serum concentration of HCV core protein measured by the sandwich FEIA (Core-Ag). Also, HCV serotypes were determined by the enzyme-linked immunosorbent assay. Testing 105 consecutive HCV-RNA positive samples showed that the PCR was the most sensitive assay(100% quantifiable), followed by the Core-Ag(82.4%) and the b-probe(75.3%). The values obtained by each method correlated well in serotype 1, whereas some discordance was noted in serotype 2 cases. Also, serotype 2 samples were less quantifiable particularly by b-probe assay than serotype 1 samples. These points have to be considered in quantification of serum HCV levels by currently available these three methods.

Pituitary adenylate cyclase-activating polypeptide induces gene expression of the catecholamine synthesizing enzymes, tyrosine hydroxylase and dopamine beta hydroxylase, through 3',5'-cyclic adenosine monophosphate- and protein kinase C-dependent mechanisms in cultured porcine adrenal medullary chromaffin cells.

Pituitary adenylate cyclase-activating polypeptide (PACAP)i a potent stimulant of catecholamine secretion, increased catecholamine production in cultured porcine adrenal medullary chromaffin cells. PACAP induced dose-and time-dependent increases in mRNAs for the catecholamine synthesizing enzymes, tyrosine hydroxylase (TH) and dopamine beta-hydroxylase (DBH), with maximal 6- and 4-fold increases occurring at 8-16 h, respectively. The half-maximally and maximally effective PACAP concentrations for stimulation of TH and DBH gene expression were 0.5 and 3 nM, respectively. The TH protein level also showed an increase over the unstimulated basal level at 16-24 h in PACAP-stimulate cells. We previously demonstrated that PACAP activates both phospholipase C and adenylate cyclase in adrenal medullary cells. Addition of forskolin alone induced increases in mRNA expression of both TH and DBH. The phosphodiesterase inhibitor 3- isobutyl-1-methylxanthine potentiated the induction of TH and DBH mRNAs by PACAP. Addition of the protein kinase C activator phorbol 12-myristate 13-acetate (PMA) also caused increases in TH and DBH mRNA levels. In protein kinase C-downregulated cells pretreated with PMA for 24 h, the stimulatory effect of PACAP on TH and DBH gene expression was diminished. These results suggest that cAMP and protein kinase C mediate the PACAP-induced TH and DBH gene expression. Removal of extracellular Ca2+ with EGTA enhanced the PACAP-induced increases in both cellular cAMP and mRNA levels of TH and DBH, suggesting that Ca2+ has an inhibitory effect on the induction of TH and DBH mRNAs. In conclusion, the present study indicates that PACAP coordinately upregulates the gene expression of both TH and DBH by activating the cAMP and protein kinase C signaling pathways, leading to simulation of cate-cholamine synthesis, while Ca2+ negatively regulates TH and DBH gene expression in porcine adrenal medullary cells.

[Expression of mRNAs coding for catecholamine synthesizing enzymes in human adrenal pheochromocytoma].

Pheochromocytomas synthesize and release catecholamines, which subsequently are related to various clinical manifestations of the disease. However, pheochromocytomas are not innervated and the catecholamine release and synthesis are not initiated by neural impulses. It is still unknown how catecholamine synthesis is regulated in pheochromocytomas. As a first step toward understanding the molecular mechanisms by which catecholamine synthesis is controlled in the tumor, we measured the levels of mRNA coding for the catecholamine synthesizing enzyme, tyrosine hydroxylase (TH) and catecholamines in 6 pheochromocytomas and 2 normal adrenal glands. The TH mRNA level was overexpressed and the catecholamine contents were high in 4 out of 6 pheochromocytomas. There was a close correlation between the TH mRNA level and the catecholamines content in the tumors. We also examined the gene expression of the messengers of other catecholamine synthesizing enzymes, dopamine beta-hydroxylase (DBH) and aromatic 1-amino acid decarboxylase (AADC) in pheochromocytomas. The expression of these genes was in parallel with that of TH mRNA in the tumors. These findings indicate that catecholamine overproduction in pheochromocytomas is mediated by the overexpression of genes coding for catecholamines synthesizing enzymes, TH, DBH, and AADC.