DNA and Cell Biology

We have constructed an improved DNA expression vector based on the Sindbis virus. Several DNA-based Sindbis virus vectors were constructed to investigate the efficiency of transgene expression. These vectors, when transfected into mammalian cells, have been used to express heterologous genes. A recombinant genome of Sindbis plasmid DNA, in which the structural genes were replaced by a polylinker cassette to allow for insertion of heterologous genes, was placed under the control of a simian virus (SV 40) promoter with a hepatitis delta virus (HDV) antigenomic ribozyme and a polyadenylation signal. Transfection of mammalian cells with this Sindbis-based plasmid vector, pSin-SV40-HDV-SV40pA, resulted in transient high-level expression of the β-galactosidase reporter gene. The expression level of β-galactosidase from pSin-SV40-HDV-SV40pA was more than 16-fold higher than that of pSin-Lux originally reported by Herweijer et al. In vivo expression was also detected after injection of plasmid DNA into mouse quadriceps. In vivo expression was transient and undetectable after day 14. Furthermore, we demonstrate that the transfection of cells with this Sindbis virus vector results in apoptotic death on glioma cells. We have demonstrated a high-level expression of the exogenous β-galactosidase gene from the pSin-SV40-HDV-SV40pA construct using a Sindbis replication system.

The induction of CYP2B gene expression by phenobarbital (PB) is mediated by the translocation of the constitutive androstane receptor (CAR) from the cytoplasm to the nucleus. The CAR/RXR heterodimer binds to two DR-4 sites in a complex phenobarbital responsive unit (PBRU) in the CYP2B gene. The short heterodimer partner (SHP), an orphan nuclear receptor that lacks a conventional DNA binding domain, was initially identified by its interaction with CAR. We have examined the role of SHP in CAR-mediated transactivation of the CYP2B gene. Coexpression of SHP inhibited the transactivation of the CYP2B gene by CAR in cultured hepatoma cells and the p160 coactivator GRIP1 reversed the inhibition. The interaction of CAR with SHP was confirmed by GST pulldown experiments. SHP did not block the binding of either CAR/RXR to the PBRU or binding of GRIP1 to the CAR/RXR complex in gel mobility shift assays, but slightly increased CAR/RXR binding and slightly altered the mobility of the CAR/RXR/GRIP1 complex, suggesting an interaction of SHP with these complexes. The presence of SHP in the complexes, however, could not be detected in an antibody supershift assay. Recombinant corepressors mSin3A, SMRT, and HDAC1, but not NCoR1, interacted with GST-SHP but each of these corepressors in liver nuclear extracts bound to GST-SHP. SMRT and NCoR1 inhibited CAR-mediated activation independent of SHP, but mSin3A and HDAC1 had little effect alone, and were additive with SHP. These studies demonstrate that SHP does not inhibit CAR-mediated trans-activation by interfering with DNA binding or by competition with GRIP1. Instead, SHP may either inhibit recruitment of other coactivators by GRIP1 or actively recruit corepressors directly to the CAR/RXR/PBRU complex.

DNA vaccines have advantages over other types of vaccines in that they can induce strong cellular immune responses, namely cytotoxic T lymphocytes (CTL) and helper T lymphocytes (Th). DNA vaccines are therefore considered a promising alternative to attenuated live vaccines in the field of infectious diseases. So far, various DNA vaccines have been generated and tried to induce a particular cellular immune response by virtue of recombinant DNA technology. DNA vaccines have been designed for efficient transcription and translation of target genes by a variety of strategies. Also, various DNA vaccine strategies for induction of specific CTL and Th have been reported by taking into consideration antigen presentation pathways and the strategies have been shown to be effective to elicit particular T-cell responses. In this paper, we have reviewed these strategies, including our study on epitope-specific T-cell induction by DNA vaccination against Listeria monocytogenes infection. From this review, it has been surmised that, to induce strong immune responses by DNA vaccines, the immunization route and the immunization regimen, such as heterologous "prime-boost" regimen, should also be considered.

In an effort to develop a more effective genetic immunization strategy for HIV, we developed an HIV-2 env DNA vaccine and evaluated three adjuvant formulations. The gp140 gene from HIV-2_UC2was synthesized using mammalian codons and cloned into a plasmid vector that expresses eukaryotic genes at high levels. We found that after three immunizations in mice, a novel cationic liposome formulation(Vaxfectin) was superior at inducing systemic and mucosal antibody responses compared to a naked DNA, a controlled release device (an Alzet minipump) and polysaccharide microparticles made from chitosan(P = 0.027). Vaxfectin also induced higher levels of systemic antibodies for each isotype and IgG subclass as well as levels of HIV-2-specific mucosal IgA (P = 0.034). When different routes of immunization were used with the Vaxfectin formulation, gp140-specific systemic antibody responses were highest by the intradermal route, mucosal antibody responses were highest by the intramuscular route, while the intranasal route was the least effective. These results suggest that this cationic liposome formulation is an important adjuvant to improve the effectiveness of genetic immunization strategies for AIDS, and that multiple routes of immunization should be employed for optimal efficacy for HIV vaccine candidates.

Lectin-like oxidized low-density lipoprotein receptor-1 (LOX-1) is a unique scavenger receptor that plays important roles in atherogenesis and has been thought to function as a monomer. Using coimmunoprecipitation studies, we demonstrate that human LOX-1 (hLOX-1) forms constitutive homo-interactions in vivo. Western blot analysis of cell lysates under nonreducing or reducing conditions revealed one clear immunoreactive species corresponding to the size of a putative receptor dimer or a monomer, respectively, consistent with the presence of disulfide-linked hLOX-1 complexes. Site-directed mutagenesis studies indicated that cysteine 140 has a key role in the formation of these disulfide-linked hLOX-1 dimers. Eliminating this intermolecular disulfide bond markedly impairs the recognition of Escherichia coli by hLOX-1. Furthermore, these dimers can act as a "structural unit" to form noncovalently associated oligomers, as demonstrated by a membrane-impermeant crosslinker, which resulted in immunoreactive species corresponding to the sizes of putative tetramers and hexamers. These results provide the first evidence for the existence of hLOX-1 dimers/oligomers.

Human papillomavirus (HPV) is implicated as an etiologic agent in neoplasitc lesions of the cervix. In this study, we used an HPV DNA chip to detect the type-specific sequence of HPV from cervical swabs in women with biopsy- proven neoplastic lesions of the cervix. Four hundred seventy-one patients were involved and classified into four groups based on the cytopathologic diagnosis: group I (normal, n= 290), group II (low-grade squamous intraepithelial lesions (SIL), n = 68), group III (high-grade SIL, n = 51), and group IV (invasive cervical cancer, n = 55). HPV detection rates were 17.6% (51 of 290), 73.5% (50 of 68), 92.2% (47 of 51), and 95.2% (59 of 62) in patients of group I to group IV, respectively. HPV-16 was the most frequent type (21.8%) in all specimens tested, and significantly increased the prevalence by advancing the grade of the cervical lesions (P < 0.01). The next frequent virus types were HPV-18 and HPV-58. The prevalence of multiple HPV infections was 37.3, 43.7, 27.7, and 28.8%, and no significant difference was detected between each group (P > 0.05). This suggests that the HPV DNA chip is a sensitive diagnostic tool for the detection of HPV in cervical specimens, and that it would provide more useful information on viral genotype and multiple HPV infections. Taken together, molecular biological data on HPV might be beneficial for the prevention and management of cervical neoplastic lesions.

Spiroplasma kunkelii is a helical, wall-less bacterium that causes corn stunt disease. In adaptation to its phloem-inhabiting parasitic lifestyle, the bacterium has undergone a reductive evolutionary process and, as a result, possesses a compact genome with a gene set approaching the minimal complement necessary for multiplication and pathogenesis. We cloned a much-reduced cell division gene cluster from S. kunkelii and functionally characterized the key division gene, ftsZ_sk . The 1236-bp open reading frame of ftsZ_sk is capable of encoding a protein with a calculated molecular mass of 44.1 kDa. Protein sequence alignment revealed that FtsZ_sk is remarkably similar to FtsZ proteins from other eubacteria, and possesses the conserved GTP-binding and hydrolyzing motifs. We demonstrated that overexpression of ftsZ_sk in Escherichia coli causes transgression of the host cell division, resulting in a filamentous phenotype. We also report, for the first time, the presence of a ftsA gene in the cell division cluster of a mollicute species.