Expression of Human Papillomavirus Type 16, Prototype and Natural Variant E7 Proteins using Baculovirus Expression System

Human papillomavirus (HPV) 16, E7 proteins derived from the prototype (Bac73) and natural variant (Bac101) E7 open reading frame were produced in Sf9 insect cells. The variant E7 gene occurred naturally by substitution mutation at the position of 88 nucleotide, resulting serine instead of asparagine. Using E7 specific monoclonal antibody (VD6), both E7 proteins were identified in recombinant baculovirus infected SF9 cells. Radiolabelling and immunoprecipitation analysis revealed that both E7 proteins were phosphoproteins. Immunostaining result showed that E7 proteins were mainly localized in the cytoplasm. Nuclear form of E7 proteins was also detected after a sequential fractionation procedure for removing chromatin structure. Considering that the VD6 recognition site in E7 protein is located within 10 amino acid at the N-terminus, this region appears to be blocked by the nuclear component. Western blot analysis revealed that nuclear form was more abundant than cytoplasmic E7 proteins. Time course immunostaining showed that the primary location of E7 protein was the nucleus and exported to the cytoplasm as proteins were accumulated. These events occurred similarly in both Bac73 and Bac101 infected Sf9 cells, suggesting that these two proteins may have similar biological functions.

Infection Rate and Serotype Distribution of Orientia tsutsugamushi arnong Field Rodents in Chollanamdo using Nested Polymerase Chain Reaction

The 86 strains of field rodents captured in Chollanamdo were analyzed its infection rates of Orientia tsutsugamushi by nested PCR. The detection rate of O. tsutsugamushi was 14.3 % in A. agrarius whereas O. tsutsugamushi could not be detected in C. lasiura. In locality, the rodents captured in the mountainous area showed higher detection rate than suburban area. In the case of detection rate by organs, the spleen was most appropriate specimen, but in two cases, O. tsutsugamushi could be detected in only kidney specimens. The major serotype of detected O. tsutsugamushi was serotype Karp, but four cases were serotype Boryong. These serotypes were confirmed by nucleotide sequence determination of amplified PCR products. In conclusion, the serotype Karp was the major O. tsutsugamushi in Chollanamdo.

Effects of several cytokines on the functions of fetal rat osteoblast-like cells in vitro / 대한치과교정학회지

Effects of several cytokines(IL -1beta, TNFalpha, and IFNgamma) have been examined on fetal rat osteoblast-like cells. To investigate whether cytokines play direct causal roles in production of lysosomal enzyme, fetal rat osteoblast-like cells were treated with IL-1beta, TNFalpha, and IFNgamma, respectively or combined. And acid phosphatase was determined by biochemical method. Alkaline phosphatase was assayed to determine the effects of IL-1beta, TNFalpha, and IFNgamma on the expression of this enzyme. And also experiment of calcified nodule formation was performed to assess the effects of cytokines on the bone-forming activity of osteoblast-like cells in vitro. Acid phosphatase activity was significantly increased by the addition of IL-1beta and TNFalpha, whereas decreased by IFNgamma, However, no significant changes in alkaline phosphatase activity was observed when the osteoblast-like cells were treated with IL-1beta and TNFalpha. Interestingly, IFNalpha showed stimulatory effect on alkaline phosphatase activity. The number of calcified nodules was decreased by treatment of cultures with 1ng/ml IL -lbeta, 20ng/ml TNFalpha, and 500 u/ml IFNgamma continuously for 21 days, while considerable number of calcified nodules were formed in control group of osteoblast-like cell in culture for 21 days. These results seem to suggest that cytokines may play crucial roles in bone remodeling through the direct action on the osteoblast-like cell.