Sung-il Lee

Construction of artificial promoters sensitively responsive to sonication in vitro

PurposeTo develop artificial promoters that are activated in response to sonication and to determine these properties in vitro.MethodsThe binding sites of four transcription factors (nuclear factor-kappa B, activating protein-1, nuclear factor-Y, and CArG element binding factor A) that are activated by oxidative stress were randomly ligated and linked to a TATA-box sequence to control the luciferase gene located downstream. Transiently transfected HeLa cells from human cervical cancer with a plasmid vector containing such a gene cassette were exposed to sonication, and enhancement of luciferase expression was assessed by dual luciferase assay.ResultsOf 62 promoters constructed, two promoters, designated clone 31 and clone 62 promoters, showed a more than tenfold enhancement 6 h after sonication with 1-MHz ultrasound at 1.0 W/cm2 for 60 s. These promoters were activated in a dose-dependent manner with the intensity and duration of sonication. The activation was attenuated by addition of dimethyl sulfoxide, an antioxidant, suggesting that oxidative stress was involved. The clone 31 promoter responded to each of two serial sonications. When sonicated 24 h after the first sonication, the peak of promoter enhancement was higher than that after the first sonication.ConclusionsA promoter sensitively responsive to sonication was constructed using the above method, possibly leading to the construction of a promoter of interest that could be applied for clinical use.

Construction of X-ray-inducible promoters through cis-acting element elongation and error-prone polymerase chain reaction

A promoter that is activated by ionizing radiation may be a useful tool for cancer therapy since, with such a promoter, the therapeutic gene can be expressed only in cancer tissues by irradiation. An artificially constructed promoter is advantageous as natural promoters may have physiological limitations. However, reasonably designing a promoter is hampered by shortage of information about the relationship between the structure and properties of a promoter DNA.

Enhancement of artificial promoter activity by ultrasound-induced oxidative stress.

We previously developed artificial promoters that were activated in response to X-ray irradiation. Sonication with 1.0MHz ultrasound that causes intracellular oxidative stress was found to activate some of these promoters though to lesser degrees. The most sensitive one among these promoters showed intensity- and duration-dependent activations by sonication. In addition, its activation by sonication was attenuated when N-acetyl cysteine was present, suggesting the involvement of intracellular oxidative stress in the activation mechanism. Improved promoters for sensitivity to X-ray irradiation were also found more sensitive to sonication. The most improved one showed 6.0 fold enhancement after sonication with 1.0MHz ultrasound at 1.0W/cm2 for 60s. This enhancement was also attenuated with the presence of N-acetyl cysteine. When stably transfected HeLa cells with the most sensitive promoter were transplanted on to mice and sonicated, luciferase activity by the promoter increased to 1.35 fold in average though it was not statistically significant compared to control. Although gene regulation in vivo by sonication was not clear, this is the first report on artificially constructed promoters responsive to ultrasound.

The First Detection of Brucella canis in Cattle in the Republic of Korea

Twenty mammary lymph node samples were collected from cattle on a farm in the Republic of Korea. These cattle were serologically negative for Brucella by tube agglutination test (≤1 : 50) and serum agglutination test (≤1 : 50). Out of 20 lymph node samples, two samples were positive for Brucella growth on Brucella agar as well as blood agar. Tests for urease, hydrogen sulphide and reactions against monospecific sera A and M indicated that these two isolates (No. 15 and 16) belong to the genus Brucella. Genus specific, AMOS (abortus, melitensis, ovis, suis) and Bruce‐ladder multiplex polymerase chain reaction (PCR) assays confirmed the Brucella isolates as either a B. abortus or a B. canis strain. This is the first report of the occurrence of a B. canis infection in cattle in Korea. More survey data are needed to determine whether B. canis is a significant aetiology in the cases of cattle brucellosis in Korea.

Efficacy of strain RB51 vaccine in protecting infection and vertical transmission against Brucella abortus in Sprague-Dawley rats

Immunizing animals in the wild against Brucella (B.) abortus is essential to control bovine brucellosis because cattle can get the disease through close contact with infected wildlife. The aim of this experiment was to evaluate the effectiveness of the B. abortus strain RB51 vaccine in protecting infection as well as vertical transmission in Sprague-Dawley (SD) rats against B. abortus biotype 1. Virgin female SD rats (n = 48) two months of age were divided into two groups: one group (n = 24) received RB51 vaccine intraperitoneally with 3 × 1010 colony forming units (CFU) and the other group (n = 24) was used as non-vaccinated control. Non-vaccinated and RB51-vaccinated rats were challenged with 1.5 × 109 CFU of virulent B. abortus biotype 1 six weeks after vaccination. Three weeks after challenge, all rats were bred. Verification of RB51-vaccine induced protection in SD rats was determined by bacteriological, serological and molecular screening of maternal and fetal tissues at necropsy. The RB51 vaccine elicited 81.25% protection in SD rats against infection with B. abortus biotype 1. Offspring from rats vaccinated with RB51 had a decreased (p < 0.05) prevalence of vertical transmission of B. abortus biotype 1 compared to the offspring from non-vaccinated rats (20.23% and 87.50%, respectively). This is the first report of RB51 vaccination efficacy against the vertical transmission of B. abortus in the SD rat model.

Immunoglobulin profiles in acute Brucellosis experimentally induced by Brucella canis in BALB/c mice.

This study evaluated profiles of immunoglobulin (Ig; IgA, IgG, IgG1, and IgG2a) response in experimental brucellosis induced with Brucella canis in BALB/c mice during an 8-week infection period. Six- to 8-week-old BALB/c mice (n = 36) were experimentally infected with 1 × 10(9) CFU of B. canis via the intraperitoneal route. Serial serum samples were collected from the mice at 0, 3, 7, 14, 21, 28, 35, 42, 49, and 56 days after inoculation. The sera were tested by the rapid slide agglutination test (RSAT) and 2-mercaptoethanol-RSAT and indirect enzyme-linked immunosorbent assay. Sera tested positive for B. canis by the RSAT and 2-mercaptoethanol-RSAT beginning from 7 days after inoculation until the end of the experiment. The IgA response was detected at 14 days after infection and reached peak levels at 21 days after infection. The IgG antibody responses were detected at 7 days after infection and reached the peak value at 35 days after infection. Data of our study demonstrated IgG2a-dominant responses over IgG1 during the course of infection (p > 0.05).

Tax peptide vaccine suppressed the leukemia in humanized mouse model of ATL

Cytotoxic T-lymphocyte (CTL) against HTLV-1 Tax has been demonstrated to play a vital role in controlling HTLV-1 infected cells in the infected carrier people. It was also shown that DNA immunization of mice with Tax expression vector suppressed the tumor growth of EL-4 lymphoma cells expressing Tax (EL4/Gax) through the activation of anti-Tax CTL [1]. Recently we established an HTLV-1-infected humanized mouse model (hu-NOG) to recapitulate ATL development and HTLV-1-specific immunity [2]. Here the efficacy of Tax-peptide vaccination was examined in the hu-NOG mouse model. Mixture of twelve overlapping peptides of 40-42 amino acids long encompassing whole Tax protein was inoculated subcutaneously three times weekly to hu-NOG mouse and then -irradiated HTLV-1 producing Jurkat cells were intraperitoneally injected to infect HTLV-1. While the control mice without immunization died of leukemia within two months of infection, Tax-immunization retarded the out growth of human lymphocytes after HTLV-1 infection and two out offive mice survived with alimited number of infected T-cells. IL-12 induction and the enhanced expression of Tax-specific CD8 T-cell were observed in the peripheral blood of immunized mice before infection. Furthermore, it was shown that the intranasal inoculation of Tax peptides has comparable effect on the onset of leukemia in the hu-NOG systems. These results suggest that the Tax peptide vaccination can elicit protective immunity against HTLV-1 infection and/or ATL developments.

Induction of APOBEC3B cytidine deaminase gene in HTLV-1 infected T-cells of ATL model mouse

A variety of mutations are accumulated in the genome of HTLV-1 infected T-cells during ATL development. To elucidate the mechanism of ATL development a mouse model of ATL was established by infecting HTLV-1 to humanized NOG mice and the infected mice recapitulate the ATL-like symptoms and die of leukemia within several months of infection. Analysis of gene expressions in the humanized mouse model of ATL demonstrated the induction of APOBEC3B (A3B) gene in the HTLV-1 infected human T-cells. A3B is a member of the APOBEC family of cellular cytidine deaminase and was recently identified as the mutational source in multiple human cancers. We have previously shown that HTLV-1 infected CD25 (-) CD4 T-cells but not CD25 (+) CD4 T-cells in ATL model mouse express a small amount of Tax mRNA even though both cell populations consist of identical infected-cell clones. As the A3B expression in HTLV-1 infected CD25 (+) T-cells was similar to, or rather higher than that in CD25 (-)T-cells, Tax appears not to be involved in the induction A3B. In contrast, ex vivo culture of infected T-cells, in which Tax expression was highly enhanced in both CD25 (+) and CD25 (-)T-cells, resulted in the suppression of A3B expression. However, ectopic expression of HTLV-1 in Jurkat cell by the transfection of HTLV-1 molecular clone plasmid enhanced the expression of A3B gene. It is thus suggested that HTLV-1 infection but not Tax induces the A3B expression. Interplay between HTLV-1 infection and A3B and the possible involvement of A3B in the mutagenesis of host cell genome are currently investigated.