Hypermodification and immune escape of an internally deleted middle-envelope (M) protein of frequent and predominant hepatitis B virus variants

Naturally occurring deletions within the human hepatitis B virus (HBV) preS2 region have frequently been identified in patients with hepatocellular carcinoma (HCC), while chronic carriers without cirrhosis and HCC contain no detectable preS2 deletion variants. We have characterized two different preS2 internal deletion variants from two patients. In addition to several weak phenotypes, our study revealed three unexpected strong phenotypes: (1) a paradoxical "hypermodification" phenomenon was observed with significantly increased size heterogeneity and molecular weights of the secreted middle (M) envelope proteins containing a preS2 internal deletion. This phenomenon was observed in transient transfection with a human hepatoma Huh7 cell line as well as in stable transfection with a rodent hepatoma cell line 7777. (2) A significantly increased intracellular accumulation of all three envelope proteins (large, middle, and small) was detected by both Western blot analysis and immunofluorescence microscopy. (3) The middle envelope proteins with a preS2 internal deletion were not recognized in vitro by a putative neutralizing antiserum, suggesting that these variants can evade immune recognition in vivo. To our knowledge, this is the first identification and characterization of the M deletion variant protein in HBV natural infection.     

Different expression of hepatitis B surface antigen between hepatocellular carcinoma and its surrounding liver tissue,studied using a tissue microarray

Hepatocellular carcinoma (HCC) is one of the most common malignancies worldwide and is highly associated with chronic liver disease, including hepatitis B viral infection. In order to study the association between hepatitis B virus (HBV) infection and HCC development, tissue microarrays were used to detect the expression of hepatitis B surface antigen (HBsAg) in 194 HCCs and their surrounding liver tissues, using anti-HBsAg monoclonal antibody. The results showed that the expression of HBsAg is significantly lower in tumour tissue than in non-tumour tissue. Among the 138 cases with positive serum HBsAg, expression of HBsAg was more frequently detected in non-tumour tissue (103 cases, 75%) than in tumour tissue (11 cases, 8%). RT-PCR and Southern blot analysis were performed to explore the mechanism of the decreased expression of HBsAg in tumour cells. The RT-PCR results showed that absence or decreased expression of the HBV S gene was detected in 3/15 (20%) and 6/15 (40%) HCCs, respectively. Integration of HBV in 23 pairs of HCCs and their matched non-tumour liver tissues was studied by Southern blot. The results showed that the integrated HBV S gene sequence was detected in 19/23 tumours (83%) and 1/23 non-tumour tissues (4%), whereas the free replicative virus form was observed in 3/23 tumours (13%) and 14/23 non-tumour tissues (61%). These findings suggest that HBsAg-negative results in tumour tissues were directly related to HBV DNA insertion and provide new insights into the involvement of HBsAg in hepatocarcinogenesis.     

Identification of a satellite double-stranded RNA in the parasitic protozoan Trichomonas vaginalis infected with T. vaginalis virus T1

Co-infection by a 0.5-kb small double-stranded (ds) RNA together with Trichomonas vaginalis virus (TVV) genomic 4.6-kb dsRNA is commonly observed in a number of T. vaginalis isolates. By molecular cloning and primer extension experiments, the 497-bp cDNA sequence of a 0.5-kb dsRNA co-infecting with TVV-T1 in T vagina/is T1 isolate was elucidated. Consistent with the replication cycle of a typical dsRNA virus, a plus-strand viral RNA beginning at +1 of the 0.5-kb dsRNA was identified in infected T. vaginalis T1 cells by primer extension and Northern hybridization studies. The 0.5-kb dsRNA was separately encased in TVV capsids from the viral genomic dsRNA, as shown by protein analysis and electron microscopic examination of viral particles purified by multiple rounds of CsCl gradient centrifugation. The riboprobes transcribed from a cloned cDNA of the 0.5-kb dsRNA exhibited strong hybridization to a small dsRNA in a T vaginalis T9 isolate, which harbors a TVV-T9 distantly related to TVV-T1, but the same probes showed very little hybridization to the viral genomic dsRNA of both TVV-T1 and TVV-T9. Very little sequence homology between the 0.5-kb dsRNA and the 4.6-kb dsRNA in TVV-T1 was found by computer-assisted analysis, suggesting that the small dsRNA in T. vaginalis T1 is not derived from the genome of TVV-T1 or other distantly related T. vaginalis viruses. These results suggest that the small dsRNAs in T vaginalis are satellite RNAs of T. vaginalis virus.     

Catechol-O-methyltransferase and Parkinson's disease

Parkinson's disease (PD) is one of the main causes of neurological disability in the elderly. Levodopa is the gold standard for treating this disease, but chronic levodopa therapy is complicated by motor fluctuation and dyskinesia. The catechol-O-methyltransferase (COMT) inhibitors represent a new class of antiparkinsonian drugs. When coadministered with levodopa/decarboxylase inhibitor, 2 COMT inhibitors, tolcapone and entacapone have been shown to improve the clinical benefit of levodopa. COMT activity is genetically polymorphic, and individuals with the low activity (COMT(L/L)) genotype have a thermolabile COMT protein; studies suggest that this genotype is less common in Asians than in Caucasians. Differences in COMT activity may determine the individual response to levodopa and result in ethnic differences in PD susceptibility. Our recent study suggests that the COMTL allele can interact with the MAOB gene to increase the occurrence of PD in Taiwanese. In order to understand this new class of antiparkinsonian drugs, we review their basic properties, pharmacology, and clinical efficacy. The frequency distribution of COMT genetic polymorphisms among different populations and its implications in the etiology and drug response is also discussed.     

Polymorphisms at the SRBI locus are associated with lipoprotein levels in subjects with heterozygous familial hypercholesterolemia

Scavenger receptor, class B, type 1 (SRBI) is a promising candidate gene involved in the pathophysiology of atherosclerosis. We have examined the association of three common polymorphisms at the SRBI locus in 77 subjects who were heterozygous for familial hypercholesterolemia (FH). The alleles represented by polymorphisms in exon 1 and exon 8 were associated with variation in plasma concentrations of fasting triglyceride (TG). Mean plasma TG concentrations for homozygotes for the most common allele, and for heterozygotes and homozygotes for the less common allele were 85 +/- 6, 111 +/- 9 and 135 +/- 22 mg/dl (p = 0.011) for exon 1, and 96 +/- 11, 86 +/- 6 and 134 +/- 13 mg/dl (p = 0.007) for exon 8, after adjustment for age, sex and body mass index. In addition, the exon 8 polymorphism was associated with increased total cholesterol (320 +/- 15, 340 +/- 8 and 388 +/- 18 mg/dl, p = 0.015), very low density lipoprotein (VLDL) cholesterol (18 +/- 2.9, 15.7 +/- 1.6 and 33.4 +/- 3.9 mg/dl, p < 0.001) and low density lipoprotein (LDL) cholesterol (251 +/- 15, 270 +/- 8 and 312 +/- 10 mg/dl, p = 0.041) concentrations. In agreement with animal studies, our data also suggest a role for the SRBI in the metabolism of apolipoprotein B (apoB)-containing lipoproteins in humans. This pathway may constitute a backup mechanism to LDL receptor-mediated pathways for the catabolism of these lipoproteins, which could be particularly relevant in subjects with high levels of apoB-containing lipoproteins, such as those occurring in patients with FH.     

PKC- and PI3K-dependent but ERK-independent proliferation of murine splenic B cells stimulated by chondroitin sulfate B

High molecular weight polyanions such as dextran sulfate are known to be weak polyclonal activators of murine B cells, but the molecular mechanism of their mitogenic activitiy is not fully elucidated. Although chondroitin sulfate A (CSA), B (CSB) and C (CSC) are highly charged polyanions, little is known about their effects on the proliferation of B cells. In this study, we demonstrated that CSB stimulated proliferation of murine B cells as markedly as did anti-IgM antibody, more markedly than did dextran sulfate and much more markedly than did CSA, CSC, heparin and hyaluronic acid. CSB caused translocation of protein kinase C (PKC) isoform beta from cytosol to membrane fractions and increased phosphorylation of Akt but not phosphorylation of extracellular signal-regulated kinase (ERK) of B cells. CSB-induced B cell proliferation was almost completely blocked by either the phosphatidylinositol 3-kinase (PI3K) inhibitor LY294002 or the PKC inhibitor GF109203X but was not significantly inhibited by the ERK kinase inhibitor PD98059. The mitogenic effect of anti-IgM was significantly inhibited by all the three inhibitors, while the mitogenic effect of LPS was inhibited only by LY294002. These findings indicate that CSB stimulated proliferation of murine B cells more markedly than did dextran sulfate and suggest that PKC and PI3K are crucial but that ERK is less important for the mitogenic activity of CSB, the signaling pathways of which may be at least partly distinct from those of anti-IgM and LPS.