Complementary DNA sequences of the constant regions of T-cell antigen receptors α, ß and γ in mandarin fish, Siniperca chuatsi Basilewsky, and their transcriptional changes after stimulation with Flavobacterium columnare.

In this study, the constant-region genes (Cα, Cß and Cγ) that encode the T-cell antigen receptor (TCR) α, ß and γ chains were cloned from mandarin fish, Siniperca chuatsi Basilewsky, an important freshwater fish species in China. The complementary DNA sequences of Cα, Cß and Cγ were 843, 716 and 906 base pairs (bp) in length and had a 465-, 289- and 360-bp 3' untranslated region, encoding 125, 142 and 182 amino acids, respectively. The amino-acid sequences of the constant regions of mandarin fish TCR α, ß and γ chains (encoded by Cα, Cß and Cγ, respectively) were most similar to those of their teleost counterparts, showing 60% similarity with pufferfish, 48% similarity with Atlantic salmon and 57% similarity with flounder, respectively. The phylogenetic analysis revealed that the mandarin fish Cα, Cß and Cγ were clustered, respectively, with their vertebrate counterparts. The mandarin fish Cα, Cß and Cγ could also be separated into four domains: immunoglobulin; connecting peptide (CP); transmembrane (TM); and cytoplasmic tail. Several conserved features in mammalian TCRs were also found in those of mandarin fish, such as a conserved cysteine residue in the CP domain of Cα, necessary for creating an interchain disulphide bond with the TCR ß chain, and a conserved antigen receptor TM motif in Cα and Cß. Meanwhile, transcripts of Cα, Cß and Cγ were detectable in all examined organs, with a stronger signal observed in lymphoid organs. In addition, the temporal transcriptional changes for Cα and Cγ were investigated, 1, 2, 3, 4, 5, 6 and 8 weeks after stimulation with Flavobacterium columnare, in head kidney, spleen, blood, thymus, gill and intestine, using real-time polymerase chain reaction. The results demonstrated stimulation-dependent up-regulations in almost all tissues examined, which indicates that T cells may play important roles in preventing mandarin fish from bacterial invasion. In particular, apart from thymus, T cells were distributed mainly in gill and intestine, where striking up-regulation of Cγ was also observed. These results will facilitate functional studies of teleost TCRs and T cells.

Immunoglobulin joining (J) chain and its expression in the Chinese soft-shelled turtle (Pelodiscus sinensis).

The immunoglobulin (Ig) joining (J) chain plays an important role in the formation of polymeric Igs and their transport into secretions. In the present study, the cDNA sequence of J chain has been cloned from the Chinese soft-shelled turtle (Pelodiscus sinensis) by reverse transcription (RT)-PCR and rapid amplification of cDNA ends (RACE). The cDNA sequence is 2347 bp in length and contains an open reading frame of 480 bp encoding 160 aa including the signal sequence. The deduced amino acid sequence has a high degree of homology with that of an already reported turtle J chain (80.7%), and of chicken (71.3%). By using real-time quantitative RT-PCR analysis, a significant up-regulation of J-chain transcripts was observed in spleen, kidney and blood of turtles injected with inactivated Aeromonas hydrophila, indicating the immune role of J chain in response to bacterial infection.

Comparative study and expression analysis of the interferon gamma gene locus cytokines in Xenopus tropicalis.

Using bioinformatics approach, the genome locus containing interleukin (IL)-22, IL-26, and interferon gamma (IFN-gamma) genes has been identified in the amphibian, Xenopus tropicalis. Like that in other vertebrates such as fish, birds, and mammals, the Xenopus IL-22, IL-26, and IFN-gamma are clustered in the same chromosome and the adjacent genes are conserved. The genomic structures of the Xenopus IL-22, IL-26, and IFN-gamma gene were identical to that of their mammalian counterparts. The Xenopus IL-22 and IL-26 genes contained five exons and four introns while the Xenopus IFN-gamma gene consisted of four exons and three introns. The Xenopus IL-22, IL-26, and IFN-gamma share 14.1-41.6%, 14.6-31.2%, and 23.7-36.5% identity to their counterparts in other species, respectively. Reverse-transcription polymerase chain reaction (PCR) and real-time quantitative PCR analyses revealed that the expression of IL-22, IL-26, and IFN-gamma genes was significantly upregulated after simulation with bacterial polyliposaccharide and/or synthetic double-stranded poly(I:C), suggesting these cytokines like those in other vertebrates play an important role in regulating immune response in Xenopus.

Mitogen-activated protein kinase signalling pathways triggered by the hepatitis C virus envelope protein E2: implications for the prevention of infection.

OBJECTIVE: Hepatitis C virus (HCV) is a major pathogenic factor of liver diseases. During HCV infection, interaction of the envelope protein E2 of the virion, with target cells, is a crucial process for viral penetration into the cell and its propagation. We speculate that such interaction may trigger early signalling events required for HCV infection. MATERIALS AND METHODS: Human liver cell line L-02 was treated with HCV E2. The kinase phosphorylation levels of mitogen-activated protein kinase (MAPK) signalling pathways in the treated cells were analyzed by Western blotting. The proliferation of the E2-treated cells was evaluated by MTT assay. RESULTS: HCV E2 was shown to be an efficient activator for MAPK pathways. Levels of phosphorylation of upstream kinases Raf-1 and MEK1/2 were seen to be elevated following E2 treatment and similarly, phosphorylation levels of downstream kinases MAPK/ERK and p38 MAPK also increased in response to E2 treatment, and specificity of kinase activation by E2 was confirmed. E2-induced MAPK/ERK activation was inhibited by the MEK1/2 inhibitor U0126 in a concentration-dependent manner. Blockage of relevant cellular receptors reduced activation of Raf-1, MEK1/2, MAPK/ERK and p38 MAPK by E2, indicating efflux of the E2 signal from extracellular to the intracellular spaces. Thus, kinase cascades of MAPK pathways were continuously affected by E2 presence. Moreover, enhancement of cell proliferation by E2 appeared to be associated with the dynamic phosphorylation of MAPK/ERK and p38 MAPK. CONCLUSION: These results suggest that MAPK signalling pathways triggered by E2 may be a potential target for prevention of HCV infection.

Inhibition of hepatitis C virus replication and expression by small interfering RNA targeting host cellular genes.

Small interfering RNA (siRNA) is a powerful tool for functional genomics and gene therapy. Viral replication and gene expression are strongly inhibited by siRNA treatment of infected mammalian cells. However, the high sequence specificity of siRNAs, combined with prolonged treatment, promote the emergence of siRNA-resistant virus variants, especially among viruses that encode a polymerase lacking proofreading capabilities, indicating that the antiviral properties of specific siRNAs are not as effective as expected. To investigate the silencing effect of siRNAs against selected host cellular proteins that promote replication of hepatitis C virus (HCV), several siRNAs against human VAMP-associated protein (hVAP-A), La antigen and polypyrimidine-tract-binding protein (PTB) were evaluated. The data show that several siRNAs markedly decreased the expression levels of corresponding cellular genes that inhibited HCV replication in Huh-7 cells. These treatments were also shown to have no impact upon cell viability. These findings provide an alternative approach for blocking HCV replication. Hence, combination therapies with siRNAs against both the virus and host genes that support virus replication are likely to be a potent approach in the treatment of chronic hepatitis C.

Selection of a phage-displayed peptide recognized by monoclonal antibody directed blocking the site of hepatitis C virus E2 for human CD81.

The human CD81 (hCD81) molecule has been identified as a putative receptor for hepatitis C virus (HCV). HCV envelope glycoprotein 2 (E2) most likely plays a pivotal role in binding to host cells by interacting with the hCD81 molecule. In this study, a phage-displayed peptide library was used to select small peptides with anti-hCD81 monoclonal antibody JS-81. The output/input ratio of phages increased about 91 fold after the third round of selection. Eight of the 30 phage clones selected from the phage library showed specific binding to the anti-hCD81 by enzyme linked immunosorbent assay (ELISA). Competitive inhibition test further demonstrated that HCV E2 could significantly inhibit the binding of a positive phage clone to anti-hCD81 JS-81. Exogenous small peptide ATWVCGPCT contained by the positive phage clones showed aligned with the hCD81 sequence from 153-161 by sequence analyses. These results suggest that the selected ATWVCGPCT is a novel hCD81-like small peptide, which can block the binding site of HCV E2 for hCD81. It may be of further application on development of antiviral agents targeting the stage of HCV entry.

Identification and expression of human CD81 gene on murine NIH/3T3 cell membrane.

The human CD81 (hCD81) molecule has been identified as a putative receptor for hepatitis C virus (HCV). In this study, eukaryotic expression vector pCDM8-hCD81 containing hCD81 cDNA and pSV2neo helper plasmid was used to cotransfect with lipofectamine into murine fibroblast cell line NIH/3T3 to establish an hCD81-expressing cell line. Resistant cell clones were obtained 20 days after the selection with neomycin (600 micro/ml) and then cultured as monoclones. The expression of the transfected hCD81 gene in the cells was verified by RT-PCR and flow cytometry analyses. One of the selected cell clones showed obvious expression of hCD81 and was named NIH/3T3-hCD81. Competitive inhibition tests indicated that the binding of monoclonal anti-hCD81 (JS-81) to NIH/3T3-hCD81 cells was inhibited by recombinant HCV E2 protein, suggesting that the expressed hCD81 molecules on NIH/3T3-hCD81 cells maintain natural conformation of binding to HCV E2. The transfected NIH/3T3-hCD81 cells should be of great potential value in studies on HCV attachment and onset of infection.

Stem-loop structures II-IV of the 5' untranslated sequences are required for the expression of the full-length hepatitis C virus genome.

The 5' and 3' untranslated regions (UTR) of the hepatitis C virus (HCV) genome contain stem-loop structures, which are important in viral gene expression and replication. In this study, the functional roles of the predicted stem-loop structures of HCV 5' UTR and 3' UTR in viral gene expression were examined using a chimeric clone of full-length HCV genomic cDNA clone and the gene for green fluorescent protein (GFP). High level expression of the HCV-GFP chimera in Huh-7 cells was accomplished by using a replication defective adenovirus that expresses T7 RNA polymerase and transcription plasmid containing full-length HCV-GFP chimera under the control of a T7 promoter. The HCV-GFP clone, with deletion of stem-loop I, expressed proteins in transfected Huh-7 cells at comparable levels to the wild type HCV clone. Other mutations of the 5' UTR, which either deleted or altered the base pairing of stem-loops II to IV, completely abolished the expression of HCV-GFP chimera. In contrast, deletion of 3' UTR sequences had no effect on HCV protein expression. These findings suggest that the stem-loop structures II to IV of HCV 5' UTR are necessary for protein expression, but that stem loop I is dispensable for protein translation. The stem-loop structures of 3' UTR of HCV genome appear to have no direct role in viral gene expression.

[Enhancement of immune responses of hepatitis B virus core DNA vaccine by a signal Peptide and a universal helper T lymphocyte epitope].

The human interleukin-2 signal peptide and a potent universal helper T lymphocyte epitope PADRE were spliced to the 5' terminus of hepatitis B viru score HBcAg gene. The modified HBcAg gene was used to construct a DNA vaccine. After the resulted DNA vaccine construct was transfected into COS7 cells, secreted HBcAg was detected in the supernatant by ELISA. BALB/c mice were vaccinated intramuscularly with the modified HBcAg DNA vaccine and the wild-type one. Serum antibodies,T lymphocyte proliferative response and cytotoxic T lymphocyte response of the immunized mice were measured. The results showed that the modified DNA construct induced cellular and humoral immune responses much stronger in vivo than the natural one did, indicating the potential value as a therapeutic vaccine for treatment of chronic hepatitis B.

Construction and identification of a single stranded cDNA clone containing full-length genome of hepatitis G virus.

AIM: To construct a single cDNA clone with full-length genome of hepatitis G virus (HGV) could be transcribed and expressed in vitro. METHODS: The 5 initial HGV cDNA fragments of Iw5, Iwq2, Iwh6, Iw3 and Iw3 used in this study were amplified from serum of a Japanese non A-E hepatitis patient. These fragments overlapped and covered the entire genome from 5'-end to 3'-end of HGV cDNA. Overlap extension PCR and ligation methods were used with 12 primers for the construction of a full-length genomic HGV cDNA clone from the subgenomic fragments. RESULTS: A single HGV cDNA clone (pHGVqz) was successfully constructed, physical mapping of the generated pHGVqz found identical to what we expected, and the sequence was deposited with the GenBank under the Accession number AF081782. The analysis of the full-length sequence, which was able to be in vitro transcribed and expressed, showed that this single clone contained 9373 nucleotides (encoding 2873 amino acids), and shared high homologies with other compared HGV isolates. CONCLUSION: A full-length genomic HGV cDNA clone is generated for the first of the kind in this study, it could be expressed and transcripted. This single cDNA clone is expected to be of importance in the investigation on replication and pathogenicity of HGV.

Genomic sequence and expression analyses of human chromatin assembly factor 1 p150 gene.

Chromatin assembly factor-1 (CAF-1) plays essential roles in eukaryotic chromatin assembly during DNA replication (Smith and Stillman, 1989. Cell 58, 15-25), (Krude, 1999. Eur. J. Biochem. 263, 1-5). Its p150 subunit, involved in interaction with histone H3 and H4, is critical to the CAF-1 nucleosome assembly activity. In this study, we sequenced a 96-kb genomic DNA region that includes a 42.8-kb CAF-1 p150 subunit gene (CHAF1A), and a 41.1-kb EEN gene. A scripted bioinformatics analysis pipeline (research agent) has been set up to annotate the BAC sequence with a set of integrated algorithms. The CAF-1 p150 subunit gene contains 15 exons and 14 introns. The promoter region is characterized by deletional analyses, revealing a potential repressor. Tissue-correlated alternative splicing forms of the transcript was initially identified by EST clustering analysis, then confirmed by RT-PCR which resulted more splicing forms than computational prediction.

Activation of Intracellular MAPK/ERK Initiated by Hepatitis C Virus Envelope Protein E2 in HepG2 Cells.

CD81, widely expressed on the surface of various human cells including hepatocytes, is a protein involved in intracellular signal transduction pathways. Recent studies suggested that human CD81 could specifically interact with hepatitis C virus (HCV) envelope protein E2. Therefore, CD81 has been identified as a putative cellular receptor for HCV. The HCV E2-CD81 interaction was considered a molecular mechanism contributing to HCV infection and pathogenicity. MAPK/ERK is characteristically associated with cell proliferation and hypertrophy. To investigate the effect of HCV on MAPK/ERK, human HepG2 cells were used in this study. CD81 expression on HepG2 cell surface was determined by flow cytometry with method of immunofluorescence. The cells were cultured in DMEM medium without fetal calf serum for 7 h, and then treated with HCV E2 protein at different time courses. Activation of MAPK/ERK in the cells was measured by Western blot, immunohistochemical and immunofluorescent analyses. Phosphorylation of MAPK/ERK was related to the concentration of HCV E2 proteins and to the time length of stimulation. MAPK/ERK in HepG2 cells was activated by HCV E2 protein, suggesting that HCV E2-CD81 interaction might be involved in intracellular signal transduction and might play an active role in HCV pathogenicity.

Construction of retroviral vector carrying HSV-tk gene under control of human AFP enhancer core sequence and human pgk promotor.

AIM: To construct retroviral vector bringing HSV-tk gene under control of human AFP enhancer core sequence and human pgk promoter. METHODS: Internal SV40 promoter was deleted by SalI from retroviral vector pMNSM to construct pMNM. HSV-tk gene driven by pgk promoter was released by BamH I from an eukaryotic expression vector pBPGK-tk, and inserted into polylinker site of pMNM to construct pMNP-tk retroviral vector. Human α-fetoprotein gene enhancer core sequence was released by EcoR I from pGEM. 7Z-AFPe plasmid was inserted into the immediate upstream of pgk promoter of pMNP-tk vector. Construction of hepatoma specific retroviral vector pMNAP-tk was completed. RESULTS: The structure of pMNP-tk and pMNAP-tk vector was confirmed by restriction analysis. CONCLUSION: The vector is of great significance for hepatoma specific prodrug transformation gene therapy.

Construction of retroviral vectors to induce a strong expression of human class interferon gene in human hepatocellular carcinoma cells in vitro.

AIM: To establish the hepatoma cell-specific expression of human interferon (IFN) gene mediated by retroviral vectors METHODS: Human interferon α and interferon ß complementary DNA (IFN cDNA) were cloned into the polylinker site of pMNSM retroviral vector to construct recombinant retroviral vectors pMNSIFNA and pMNSIFNB, with the transcription of IFN gene being driven by Simian virus 40 early region promoter (SV40) early region promoter. IFN cDNAs were also cloned into pMNAIFNA, pAMNSIFNA, and pMNAIFNB, with the transcription of IFN gene being driven by SV40 early region promoter regulated by α-fetoprotein enhancer. Next, the retroviral constructs were introduced into retroviral amphotropic packaging cells using the lipofectamine-mediated gene transfer procedure. The rate of plasmid transfection was (4-40) × 10(3) colonies/µg DNA/10(6) PA317 cells. The rate of retrovirus infection was (5-500) × 10(4) colony forming units (CFU)/mL. Further, the recombinant retroviruses were used to infect human hepatoma cells, renal carcinoma cells, and melanoma cell lines in the presence of 4 µmg/L polybrene. RESULTS: Northern and Dot hybridization of total RNA from the neomycin-resistant colonies and IFN expression assay indicated that human α fetoprotein enhancer induced efficient and specific transcription and expression of IFN genes driven by the promoter of different origins in human hepatoma cells, leading to high production of α fetoprotein. CONCLUSION: Cis active element of α-fetoprotein gene can drive specific expression of IFN genes in human hepatoma cells, which provides some valuable data for the hepatoma-specific immune gene therapy.

[Genotyping of hepatitis C virus in patients with hepatocellular carcinoma].

Forty-two patients with hepatocellular carcinoma (HCC) were examined for hepatitis C virus (HCV) RNA in liver tissues by reverse transcription-polymerase chain reaction (RT PCR). Typing of HCV liver samples of 18 patients was dependent on the amplification of NS5 region by PCR using type-specific primers. Type-II was found in 14 of the 18 patients (78%), 7 of the 18 patients (39%) and 4 of the 18 patients (22%) were positive for type-II and I and for type-II and III or IV (III/IV), respectively. Type V or VI (V/VI) infection was not observed. These data indicate that HCV type-II may be the major type in HCC patients with HCV infection in China, and some patients can be coinfected with type-II and I or III/IV.

delta-9-Tetrahydrocannabinol (THC) decreases the number of high and intermediate affinity IL-2 receptors of the IL-2 dependent cell line NKB61A2.

Treatment of the cloned NK-cell line (NKB61A2) with the major psychoactive marijuana component, delta-9-tetrahydrocannabinol (THC), for 24 h suppressed IL-2-induced proliferation of these cells in the cytokine concentration range of 0.25-10 pM suggesting that the drug inhibits the functional activity of the high affinity IL-2R. The proliferation inhibitory effect of THC was accompanied by a decrease in the number of high and intermediate affinity IL-2 binding sites as measured by equilibrium binding studies. However, the expression of Tac protein on the surface of these cells was increased as determined by flow cytometry analysis. THC was also shown to decrease proliferation and the number of IL-2 binding sites of cells previously pulsed with IL-2 and then treated with the drug in the absence of IL-2. These results suggest that THC inhibits IL-2-induced proliferation by modulating the expression of high affinity IL-2 receptors (alpha/beta) required for cell activation and also suppresses the ongoing process of functional receptor expression and clonal expansion of cells previously activated by IL-2. Because the number of intermediate binding sites is decreased following drug treatment along with an increase in the expression of Tac protein (alpha chain), the lowering of high affinity sites possibly results from a drug-induced depression of beta chain expression.

A case of cerebral aspergillosis caused by Aspergillus nidulans. Clinical, pathologic and mycologic identifications.

A case of cerebral aspergillosis is reported, the presenting symptom was numbness of right face, which worsened after one year. CT-scan showed two enhanced low-density patches in the anterior and basal parts of right temporal lobe. During operation, an abscess in the deep part of right temporal lobe was revealed. The patient gradually felt amaurosis and oculomotor palsy of right eye. About six months later, she died from intracranial hypertension. Biopsy, as well as autopsy findings suggested fungal infection, and was identified as Aspergillus nidulans, which has probably never been reported in the literature.