Keyong Ho Lee

Inhibition of complement activation by recombinant Sh‐CRIT‐ed1 analogues

Sh‐CRIT‐ed1 is a potent anti‐complement peptide that inhibits the classical complement‐activation pathway by interfering with the formation of the C3‐convertase complex, C4b2a. C2 is an essential serum glycoprotein that provides the catalytic subunit of the C3 and C5 convertases of the classical pathways of complement activation. Because only in its C4‐bound state is C2a capable of cleaving its physiological protein substrates C3 and C5, the interaction of Sh‐CRIT‐ed1 with C2 plays a decisive role of inhibition in the classical complement‐activation process. However, the role of individual Sh‐CRIT‐ed1 amino acid residues in C2 binding is not fully understood. We constructed nine recombinant Sh‐CRIT‐ed1 (rSh1) analogues, substituted at conserved residues, and evaluated their anti‐complement and C2‐binding activities. Results from glutathione S‐transferase (GST) pull‐down and haemolytic assays suggested that residues 10K, 17E, 19K and 26Y are critical for the interaction of rSh1 with C2. We then constructed an improved anti‐complement peptide by duplicating Sh‐CRIT‐ed1 C‐terminal motifs (17H–26Y). This linear homodimer (rH17d) was more potent than rSh1 with respect to binding to C2 and anti‐complement activity (the 50% inhibitory concentration value was ≈1·2 µm versus ≈6·02 µm for rSh1). Furthermore, rH17d showed higher anti‐complement activity in vivo, providing additional evidence that this duplication is a more effective inhibitor of complement activation than rSh1. Taken together, these results identify four key residues in rSh1 and strongly suggest that rH17d is a potent inhibitor of complement activation that may have therapeutic applications.

Protective effects of fucoidan against γ-radiation-induced damage of blood cells

Fucoidan, a sulfated polysaccharide purified from brown algae including Fucus vesiculosus and Laminaria japonica, has a variety of biological activities, including antioxidant and antitumor activities. Here, we investigated the radioprotective effects of fucoidan on human monoblastic leukemia cell line U937. Further, animal tests were carried out using Balb/c mice in order to determine the radiation-induced changes in the counts of blood cells, including thrombocytes, erythrocytes, leukocytes and hematocrit. Cell viability was assessed by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay, wherein fucoidan (1, 10, and 100 μg/mL) was observed to improve recovery from damage caused by 8-Gy radiation in a dose dependent manner. The viability of U937 cells pre-treated with fucoidan also increased in a dose dependent manner. Furthermore, fucoidan at 100 mg/kg was found to protect against changes in the counts of blood cells as follows: on day 28 after irradiation, the thrombocyte count in the irradiated controls decreased to 45% compared with the non-irradiated controls, while that in the fucoidan-treated group was 60%. The hematocrit in the fucoidan-treated group recovered to 75% on day 28, while that in the irradiated control was 68%. The erythrocyte count in the irradiated controls consistently ranged from 64% to 67% throughout the experiment, but that in the fucoidan-treated group increased gradually, ranging from 75% to 80%. The mean number of survival days and 50-day actuarial survival rate increased dose dependently in the fucoidan-treated group. The mean number of survival days and the 50-day actuarial survival rate in this group was 16, 21, and 29 days and 12%, 20%, and 30% at fucoidan doses of 1, 10, and 100 mg/kg. The values of these parameters in the control group were 9 days and 0%, although the difference between the test and control groups was not statistically significant. Our results may prove valuable in the field of radioprotection.

Correlative Effect between in vivo Hollow Fiber Assay and Xenografts Assay in Drug Screening

PURPOSE This study was carried out to assess the usage of an in vivo hollow fiber assay to screen drugs with highly predictive accuracy. MATERIALS AND METHODS The assay systems used were the hollow fiber and xenografts assays. The hollow fiber assay was carried out with the following steps; preparation of fibers, preparation of cells, loading and implanting fibers, treatment with drugs, removal of fibers and assaying for the cell viability by the MTT assay. For the xenografts assay, cell suspensions were subcutaneously transplanted into the mice. Therapy was started when the tumor volume reached 100 approximately 200 mm(3). The tumor volumes were calculated using the formula V=[length+(width)(2)]/2, and used for evaluating the efficacy of the drugs. The drug treatment doses used were adriamycin 2.1 mg/kg, mitomycin-C 0.25 mg/kg, 5-fluorouracil 24.5 mg/kg and paclitaxel 2.5 mg/kg, and administrated intravenously five times daily. RESULTS The correlation between the xenografts and hollow fiber assays was evaluated in 20 tumor cell lines and 4 anti-cancer agents. In the 20 tumor cell lines, the overall predictive accuracy of the hollow fiber assay for sensitivity was 83%, with a predictive accuracy for resistance of 92%. CONCLUSION The hollow fiber assay was assessed as effective in drug efficacy evaluation, and found to be compatible with that of the xenografts assay.

Fucoidan Protects Human Skin Fibroblast Cell Line HS68 Against γ -Radiation-Induced Damage

Fucoidan is a sulfated polysaccharide purified from brown algae including, Fucus vesiculosus and Fucus vesiculosus and Laminaria japonica. It has a variety of biological effects including antioxidant and antitumor activity. In this study, we investigated the radioprotective effects of fucoidan on normal human newborn foreskin fibroblast cell line HS68. To evaluate the effects of fucoidan, we assayed cell viability in vitro and change of blood cells such as thrombo- cytes, erythrocytes, leukocytes and hematocrit with radiation. In a viability assay, fucoidan increased dose-dependently the recovery of radiation-induced damage by 8Gy at all tested dose (10, 50 and 100 μg/ml). The survival rate of HS68 cells by pre-treatment with fucoidan was increased by 2 times more than its compared with untreated cells. Furthermore, fucoidan protected the change of blood cells as follows; the thrombocyte of the irradiated controls had fallen to 35% com- pared with the initial values, the thrombocyte counts in fucoidan pre-treated group was recovered to 49% at the day 14. The Hematocrit level of fucoidan pre-treated group showed the recovery activity by 72% at the day 14, while hematocrit level of irradiated control without fucoidan fell to 61%. In case of erythrocyte level, the radiated controls was consistently maintained by the end of experiment at the range of 60~70%, on the other hand, the erythrocyte counts of fucoidan pre- treated group gently increased the level of erythrocyte at the range of 82~90%. These results may provide valuable and useful information in the field of radio-protection.