• Parasites, Hosts and Diseases
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• v.54 no.4
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• pp.385-391
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• 2016

The discovery and understanding of antigenic proteins are essential for development of a vaccine against malaria. In Plasmodium falciparum, Pf92 have been characterized as a merozoite surface protein, and this protein is expressed at the late schizont stage, but no study of Pv92, the orthologue of Pf92 in P. vivax, has been reported. Thus, the protein structure of Pv92 was analyzed, and the gene sequence was aligned with that of other Plasmodium spp. using bioinformatics tools. The recombinant Pv92 protein was expressed and purified using bacterial expression system and used for immunization of mice to gain the polyclonal antibody and for evaluation of antigenicity by protein array. Also, the antibody against Pv92 was used for subcellular analysis by immunofluorescence assay. The Pv92 protein has a signal peptide and a sexual stage s48/45 domain, and the cysteine residues at the N-terminal of Pv92 were completely conserved. The N-terminal of Pv92 was successfully expressed as soluble form using a bacterial expression system. The antibody raised against Pv92 recognized the parasites and completely merged with PvMSP1-19, indicating that Pv92 was localized on the merozoite surface. Evaluation of the human humoral immune response to Pv92 indicated moderate antigenicity, with 65% sensitivity and 95% specificity by protein array. Taken together, the merozoite surface localization and antigenicity of Pv92 implicate that it might be involved in attachment and invasion of a merozoite to a new host cell or immune evasion during invasion process.

• Asian Pacific Journal of Cancer Prevention
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• v.13 no.12
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• pp.6511-6516
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• 2012

Recently, we reported that an ethanol extract of Iris nertschinskia induces p53-dependent apoptosis in the MCF7 human breast cancer cell line. However, the detailed mechanisms were not fully explored. Here, we demonstrate another aspect of the activity of I. nertschinskia in breast cancer cells. We compared the response to an ethanol extract of I. nertschinskia in two different human breast cancer cell lines, Hs578Tand MDA-MB231, respectively with relatively low and high AKT1/2 activity by trypan blue exclusion assay and FACS analysis. Knockdown of endogenous AKT1 or AKT2 in breast cancer cells by RNA interference determined the sensitivity to I. nertschinskia ethanol extract compared to control cells. The I. nertschinskia ethanol extract induced cell death in a manner that depended on the level of phosphorylated AKT1/2 protein and was associated with a significant increase in the sub-G1 cell population, indicative of apoptosis. Our results indicate that an ethanol extract of I. nertschinskia differentially induces cell death in breast cancer cells depending on their level of phosphorylated AKT1/2.

• Asian Pacific Journal of Cancer Prevention
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• v.17 no.9
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• pp.4217-4222
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• 2016

Purpose: To compare the expression level of CK 15 in normal esophageal and esophageal squamous-cell carcinoma (ESCC) tissues and analyse possible functions of CK15 in occurrence and development. Materials and Methods: Immunohistochemistry was used to compare CK14, CK15 and proliferating cell nuclear antigen (PCNA) expression levels in ESCCs. Expression level of CK15 was also assessed by Western blotting. In addition, levels of CK15, cytokeratin 19 fragment antigen 21-1 (CYFRA21-1) and PCNA were detected in serum by enzymelinked immunosorbent assay (ELISA) and chemiluminescence methods. Relationships between clinicopathological parameters and CK14 and CK15 expression were then analyzed. Results: According to immunohistochemistry, in esophageal and intraepithelial neoplasia (SIN) tissues, the expression of CK14, CK15 and PCNA localized to basal layer of the epithelium. CK14 and CK15 levels were higher in normal esophageal squamous epithelial tissue than in SIN and ESCC, and greater in highly differentiated than poorly differentiated carcinoma tissue. By Western blotting, we found more pronounced expression of CK15 in normal esophageal tissue, compared with carcinoma tissue. The specificity of changed CK15 and CYFRA21-1 expression was respectively 90.0% and 96.7% in serum of ESCC patients. Joint detection could improve the sensitivity of esophageal carcinoma diagnosis. Relationships between CK14, CK15 expression and clinical parameters were not statistically significant (P>0.05). Postoperative survival in patients of CK14, CK15 positive expression was longer than with negative expression ($x^2=4.35$, P=0.037; $x^2=9.852$, P=0.002). Conclusions: CK15 expression decreased in esophageal squamous cell carcinoma tissue and serum of esophageal squamous carcinoma patients. We infer that CK15 may play an important role for the occurrence and development of esophageal squamous-cell carcinoma. In the future, CK15 may be used for the diagnosis, treatment and prognostic evaluation of esophageal squamous-cell carcinoma.

• Journal of Plant Biology
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• v.24 no.4
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• pp.171-179
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• 1981

The membrane-bound ATPases were separated on sucrose gradient from corn roots and characterized by pH optima, sensitivity to monovalent salt, Km and Vmax. The pH optima for the activity of all the ATPases associated with 13, 000g pellet and 13, 000~80, 000g pellet were 5 and 9, respectively. The ATPases in Fractions B and C of the 13, 000 g pellet were more active at pH 5 than pH 9. While, in the case of Fractions D, E and F, they were reverse. The activities of the ATPase in Fractions A and C of the 13, 000~80, 000 g pellet were greater at pH 5 than pH 9. On the other hand, the ATPases in Fractions B, D, E, and F were more active at pH 9 than pH 5. The optimum concentraction of ATP for the assay was about 3 to 5 mM. The Km's for the membrane-bound ATPases in 13, 000g pellet and in 13, 000~80, 000 g pellet were 0.25 mM. While Vmax values for 13, 000g pellet were from 8.0 to 12.5 $\mu$M Pi/mg protein/hr. according to pH values, those for 13, 000~80, 000 g pellet were from 35.7 to 55.6 $\mu$M Pi/mg protein/hr. Activities of the membrane-bound ATPases in both 13, 000 g pellet and 13, 000~80, 000 g pellet were stimulated with increasing the concentration of $K^+$.

• The Journal of Internal Korean Medicine
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• v.19 no.1
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• pp.221-246
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• 1998

The aim of this experimental study was to evaluate the anti-tumor effects of Chungsimbohyeltang through investigation about viability of tumor cell by MTT assay, survival period of mice transplanted with L-1210 cells, growth inhibition on the tumor cell, body weight variation in mice transplanted with sarcoma-180 cells and its immunomodulatory effects through the investigation on delayed type hyper-sensitivity, the hemagglutinin and hemolysin titers for humoral immune response, the appearance of rosette forming cells for cell-mediated immune response, the natural killer cell activity, the transformation of lymphocyte, the productivity of Interleukin-2 and phagocytic index K was performed in immune-depressed ICR mice induced by methotrexate treatments. The results were as follows ; 1. $IC_{50}$ of Chungsimbohyeltang treated group was 5.85mg/ml in SNU-C4 cell, 1.38mg/ml in SNU-396 cell, 0.21mg/ml in SNU-1 cell, so it had low anti-tumor activity. 2. The both groups of Chungsimbohyeltang extract 10mg/kg and Chungsimbohyeltang extract 20mg/kg had no toxicity and the group of Chungsimbohyeltang 20mg/kg which was shown 120% in ILS had the effect of life prolongation in mice transplanted with L-1210 cells. 3. In the growth inhibition on the tumor cells, only the group of Chungsimbohyeltang extract 20mg/kg was noted and in the weight variation in mice transplanted with sarcoma-180 cells, both groups of Chungsimbohyeltang extract had a significant effect. 4. In the delayed type hypersensitivity and appearance of rosette forming cells, both groups of Chungsimbohyeltang extract didn't have any significant effect. 5. The hemagglutinin titers was slightly increased with no significance, and the hemolysin titers was significantly increased in the only group of Chungsimbohyeltang extract 20mg/kg. 6. The natural killer cell activity of the Chungsimbohyeltang extract groups was significantly increased in the ratio of 100:1 of effector and target cells, but it was not significantly increased in the ratio of 50:1, 10:1. 7. The transformation of lymphocyte and the productivity of Interleukin-2 were increased significantly and in dose-dependent manner in both group of Chungsimbohyeltang extract. 8. The phagocytic effect of macropage was significantly increased in both groups of Chungsimbohyeltang extract. Considering the results above, we could conclude that Chungsimbohyeltang have an indirect anti-tumor effect through the modulation of immunme response, although it had not toxicity on the tumor cell it self.

• Parasites, Hosts and Diseases
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• v.24 no.2
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• pp.171-176
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• 1986

In order to obtain the most specific and sensitive antigen from crude antigens of Fasciola hepatica for the immunodiagnosis of bovine fascioliasis by the enzyme linked immunosorbent assay (ELISA), phosphate buffered saline extract of F. hepatica was prepared. The crude extract was fractionated into 7 antigens using sephadex G-100 column chromatography. Seven fractionated antigens were applied to ELISA, precipitation test and intradermal test, respectively. Results obtained are as follows: 1. The specificity (95% confidence interval in negative sera of bovine fascioliasis; Mean+$2{\times}SD$ of absorbence) of the first (MW>150,000) and the second antigens(MW 120,000) were 93.7%, but those of others including crude antigen showed 100%. 2. The sensitivity(positive sera of bovine fascioliasis having higher values with compared to the criterion) of the first, the sixth (MW 16,000) and the seventh antigen (MW<5,000) were 91.6%, 87.5% and 0%, respectively, but those of others showed all 100%. 3. The absorbance by ELISA using the fifth antigen (MW 26,000) was 8. 43-folds higher in the positive sera than that in the negative sera. This could be used as one of the most specific antigens for the immunodiagnosis of bovine fascioliasis. 4. In Ouchterlony test, precipitin lines were not found in the sera naturally infected with F. hepatica, but some were found in the sera of rabbits immunized with the crude antigens. The numbers of precipitin lines in the sera of rabbits were different in the different fractionated antigens. They were 6 in the crude, 2 in the second and the third antigens, 1 in the fourth, the fifth and the sixth antigens and absent in the seventh antigen, respectively. 5. The wheal size for bovine infected with F. hepatica was $2.46{\pm}0.15cm$ in the intradermal test antigen(saline extract of F. hepatica) supplied by the Veterinary Research Institute, Rural Development Administration, Korea. The wheal size of the first, the second and the third antigens were larger than that. of intradermal test antigen, whereas those of the fouth, the fifth, the sixth and the seventh antigens showed smaller than that of the intradermal test antigen. The results suggest that the fifth antigen may be specific antigen for the immunodiagnosis of bovine fascioliasis.

• Parasites, Hosts and Diseases
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• v.37 no.3
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• pp.163-169
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• 1999

House dust mite allergens have been well established as sensitizing agents that are important in the induction of allergic diseases. In order to analyze epitopes of the allergen and to develop a quantitative method of the allergen exposure, monoclonal antibodies against a recombinant Der p 2 (rDer p 2), one of the major allergens of Dermatophogoides pteronyssinus, were produce. Four monoclonal antibodies produced wee species-specific and did not cross-react to the D. farinae crude extract. Two of the monoclonal antibodies were found to be IgG1 and the others were IgM. For the analysis of epitopes, a Der p 2 cDNA encoding 126 amino acids (aa) was dissected into three fragments with several overlapping peptides, A (aa residues 1-49), B (44-93), and C fragment (84-126). Three monoclonal antibodies showed reactivities to the recombinant B fragment and to the full-length rDer p 2, but one monoclonal antibody reacted only with the full-length rDer p 2. Two-site capture ELISA was developed using two different monoclonal antibodies for quantitating Der p2 in house dust. The sensitivity limit was 4ng/ml with rDer p2 and $8{\;}\mu\textrm{g}/ml$ with the d. pteronyssinus crude extract. The result suggested that the assay using monoclonal antibodies against rDer p2 could be useful for the environmental studies and for the standardization of mite allergen extracts.

• Asian-Australasian Journal of Animal Sciences
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• v.16 no.9
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• pp.1254-1260
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• 2003

The purpose of this study firstly was conducted to establish a radioimmunoassay (RIA) of estrone sulfate ($E_1S$), secondly to monitor the reproductive status of dairy cows using their urine samples. Urine and blood samples were collected in series within a day from four pregnant Holstein-friesian cows to evaluate the relationship between $E_1S$ levels in blood and urine with or without urinary creatinine basis. The urine was then collected biweekly from three cows in estrous and those artificially inseminated; collection from pregnant cows was made on a monthly basis. Results indicated that sensitivity for the $E_1S$ RIA was 5 pg/tube and the recovery rate was 100%. The daily urinary creatinine concentrations fluctuated within a day, but changes were slighter in midday, whereas the changes of concentrations of $E_1S$ in urine were relatively smaller. The concentrations of serum $E_1S$ during the estrous cycle were undetectable due to the limitation of assay, but the urinary $E_1S$ level could be measured with no obvious changes during the cycle. The urinary $E_1S$ levels increased remarkably around 7.7 to 8.3 ng/ml, 80 to 100 days after pregnancy but the serum $E_1S$ levels did not elevate until 120 to 150 days. The level of $E_1S$ increased gradually during pregnancy and eventually reached its peak before parturition at around 40 ng/ml and finally decreased to its basal level 2 days postparturition. During pregnancy, $E_1S$ concentrations of urine increased earlier than those in blood. The correlation coefficients between urinary and serum $E_1S$ concentration during pregnancy and postparturm were higher than those adjusted with creatinine (creatinine ratio). The concentrations of $E_1S$ in urine could be maintained unchanged for 8 days storing the samples in room temperature, which was extended to 8 days when the samples were pretreated by boiling for 30 minutes or treated with autoclave. In conclusion urinary $E_1S$ concentrations can be used directly for monitoring the pregnant status and fetal viability of dairy cows and can assist accurate confirmation of pregnancy in cows at least 80 to 100 days after insemination much earlier than by serum $E_1S$.

• Asian Pacific Journal of Cancer Prevention
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• v.14 no.10
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• pp.6135-6140
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• 2013

Background: Breast cancer is a common malignant tumor which affects health of women and multidrug resistance (MDR) is one of the main factors leading to failure of chemotherapy. This study was conducted to establish paclitaxel-resistant breast cancer cell line and nude mice models to explore underlying mechanisms of MDR. Methods: The breast cancer drug-sensitive cell line MCF-7 (MCF-7/S) was exposed in stepwise escalating paclitaxel (TAX) to induce a resistant cell line MCF-7/TAX. Cell sensitivity to drugs and growth curves were measured by MTT assay. Changes of cell morphology and ultrastructure were examined by optical and electron microscopy. The cell cycle distribution was determined by flow cytometry. Furthermore, expression of proteins related to breast cancer occurrence and MDR was tested by immunocytochemistry. In Vivo, nude mice were injected with MCF-7/S and MCF-7/TAX cells and weights and tumor sizes were observed after paclitaxel treatment. In addition, proteins involved breast cancer and MDR were detected by immunohistochemistry. Results: Compared to MCF-7/S, MCF-7/TAX cells had a higher resistance to paclitaxel, cross-resistance and prolonged doubling time. Moreover, MCF-7/TAX showed obvious alterations of ultrastructure. Estrogen receptor (ER) expression was low in drug resistant cells and tumors while expression of human epidermal growth factor receptor 2 (HER2) and Ki-67 was up-regulated. P-glycoprotein (P-gp), lung resistance-related protein (LRP) and glutathione-S-transferase-${\pi}$ (GST-${\pi}$) involved in the MDR phenotype of resistant cells and tumors were all overexpressed. Conclusion: The underlying MDR mechanism of breast cancer may involve increased expression of P-gp, LRP and GST-${\pi}$.

• Asian Pacific Journal of Cancer Prevention
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• v.14 no.9
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• pp.5231-5235
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• 2013

Objective: NF-E2-related factor 2 (Nrf2) is activated in several human malignancies. However, the role of Nrf2 in gastric cancer (GC) remains incompletely understood. In this study, we therefore analyzed associations of Nrf2 expression status with clinical features and chemotherapeutic resistance in GC. Materials and Methods: A total of 186 samples from GC patients who underwent gastrectomy were used for prognostic assessment. A further 142 samples from GC cases who received first-line combination chemotherapy were applied for investigation of chemoresistance. The Nrf2 expression was evaluated by immunohistochemistry in GC samples, and its relationship with clinicopathological parameters and chemotherapy sensitivity was analyzed. The effect of Nrf2 gene silencing on chemotherapy resistance was also examined by cell viability assay in vivo. Results: Of the 186 patients with GC, 104/186 (55.9%) showed high expression for Nrf2. The overexpression of Nrf2 was an independent predictor of overall survival [OS, hazard ratio (HR) 3.9; P=0.011] and disease-free survival (DFS, HR 4.3; P=0.002). The gene silencing of Nrf2 reduced resistance to cell death induced by 5-FU in GC cell lines. Conclusion: Our data show that Nrf2 is an independent prognostic factor in GC. Furthermore, Nrf2 confers resistance to chemotherapeutic drug 5-FU in GC cells. Taken together, Nrf2 is a potential prognostic marker and predictive for 5-FU resistance in GC.