Current Progress of Next Generation Battery of Toxicology-Cellular and Molecular Toxicology, and Toxicogenomics

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Protective Effect of Heat Shock Protein 70 Against Oxidative Stresses in Human Corneal Fibroblasts

We evaluated DNA protection effect of heat shock protein (HSP) against cytotoxic effects of exogenous nitric oxide (NO) and reactive oxygen intermediate (ROI). Cultured human corneal fibroblasts were divided into 4 groups. Control (Group I) was not exposed to a sub-lethal heat treatment. Other 3 groups were exposed to 43 degrees C for 1 hr, then incubated at 37 degrees C during different duration (1, 6, 24 hr, Group II, III, IV, respectively). Expression pattern of HSP 70 was analyzed by Western blot. Cell viability was measured by MTT assay and the relationship between HSP 70 expression and DNA damage was examined by terminal deoxyribonucleotidyl transferase mediated dUTP-digoxigenin nick and labeling (TUNEL) stain and single cell gel electrophoresis. Expression pattern of HSP 70 was dependent on recovery times. Cell viability following heat treatment was significantly increased and the TUNEL positive cell number was decreased at 6 hr. In single cell gel electrophoresis, tail moments were increased in a dose-dependent manner by SNAP and X/XO. Following heat treatment, tail moments showed decreased significantly at 6 hr. These results suggest that induction of HSP 70 by sub-lethal heat treatment is closely related with cytoprotective effects against oxidative stresses in human corneal fibroblasts.

Antagonism of Sophoricoside from Sophorica japonica on IL-5 / 천식및알레르기

BACKGROUND: Allergic disease is an inflammatory disease, whose main inflammatory cells are eosinophils, mast cells, and T lymphocytes. From that point, new therapeutic targets on allergic inflammation focusing on them are under investigation. We extracted four isoflavonoids from Sophorica japonica such as sophi, orobol, genistin, and genistein which has been known as PTK antagonist. We documented those three iso-flavonoids except genistein had an anti-inflammatory effect as potent as dexamethasone on carageen-induced ear model. Also they had antagonism on the Y-16 cell line, whose growth is dependent on IL-5. OBJECTIVES: From above results, in this experiment, we tried to find antagonistic effects of those compounds on IL-5 using the inhibition of eosinophil activation and survival in vitro and possibility of anti-allergic medicine. METHODS: LTC4 by RIA and ECP for degranulation by UniCAP(TM), which had been used previously were activation markers. RESULTS: Among those compounds, sophi was the most potent antagonist on IL-5 induced LTC4 release, degranulation, and survival. Orobol and genistin also had antagonism on them, but genistein, an antagonist of PTK didn't show any antagonistic effects. CONCLUSION: From these results, we concluded those three iso-flavonoids were IL-5 antagonist, and the mechanism of it might not be through PTK signaling.

O-glucogenistein inhibits eosinophil recruitment and nasal allergic symptoms in a murine model of nasal allergy / 천식및알레르기

BACKGROUND: Infiltration of eosinophils in the nasal mucosa is a consistent feature of nasal allergic inflammation. Various cytokines, especially interleukin-5(IL-5), were identified to play important roles in the infiltration and activation of eosinophils in nasal mucosa. Our previous study found that among 4 kinds of sophoricosides extracted from Sophora japonica, named sophi, orobol, genistin, and genistein, 3 compounds except genistein known as protein tyrosine kinase(PTK) inhibitor had anti-inflammatory and anti-IL-5 effects, and sophi was the most potent. OBJECTIVE: The goal of this study was to investigate the antagonism of sophi on the nasal eosinophilia in ovalbumin(OA)-sensitized murine nasal allergy model. METHODS: Male BALB/c mice sensitized intraperitoneally and then topically with OA were treated with sophi(10 or 30mg/kg) or anti-mouse IL-5 monoclonal antibody(anti-IL-5 mAb, 1mg/Kg) intravenously 1 hour before challenge. The effect of sophi on the infiltration of eosinophils into the nasal mucosa, peripheral blood eosinophilia, nasal symptom, and OA-specific IgE antibody production were evaluated. Results: Administration of sophi(10, 30mg/kg) significantly inhibited the nasal eosinophil infiltration and nasal symptom compared to that of anti-IL-5 mAb. But eosinophil count inthe peripheral blood and the titer of OA-specific IgE were not affected by sophi. CONCLUSION: Sophi inhibited not only the tissue eosinophilia but also the acute nasal allergic symptom. These findings suggest that sophi has anti-eosinophilic cytokine activity and also plays blockade of early allergic reaction. Taken together, sophi may be a candidate for new anti-allergic medicine.

Antagonism of sophoricoside from sophorica japonica on GM-CSF-induced cosinophil activation / 천식및알레르기

BACKGROUND: allergic disease is an inflammatory disease, whose main inflammatory cells are eosinophils, mast cells, and T lymphocytes. From that point, new therapeutic targets on allergic inflammation focusing on them are under investigation. We extracted four isoflavonoids from sophorica japonica such as sophi, orobol, genistin and genistein which are known PTK antagonists. We documented that these iso-flavonoids except genistein had an antagonism on IL-5 and IL-3 in vitro eosinophil activation and also in allergic mouse model sensitized by OA(ovualbumin). Their common action is due to the common beta chains. GM-CSF also share common beta chains, through which it activates eosinophils. OBJECTIVES: From the above results, We observed the antagonistic effects of these compounds on GM-CSF using eosinophil activation in vitro. METHODS: LTC4 which was detected by RIA and ECP by UniCAP were activation markers. RESULTS: Among those compounds, sophi was the most potent antagonist on GM-CSF induced LTC4 release and even on degranulation and orobol and genistin also had antagonism on them but genistein an antagonist of PTK did not show any antagonistic effects. CONCLUSION: From these results, We concluded these three iso-flavonoids were GM-CSF antagonists and the mechanism might not be through PTK signaling.

Comparison of inodilator effect of higenamine, YS49, YS51, tetra-hydroisoquinoline analogs, and dobutamine in the rat

Tetrahydroisoquinoline (THI) alkaloids can be considered as cyclized derivatives of simple phenylethylamines. Many of them, especially with 6,7-disubstitution, demonstrate a relatively high affinity for catecholamines. Present study examines the pharmacological action of limited series of THI, using rats' isolated atria and aorta. In addition, a (3H) prazosin displacement binding study with THI compounds was performed, using rat brain homogenates to investigate whether these probes have a-adrenoceptor affinity. We also compared the vascular relaxation potency of these probes with dobutamine. YS 49, YS 51, higenamine and dobutamine, concentration-dependently, relaxed endothelium-denuded rat thoracic aorta precontracted with phenylephrine (PE, 0.1 micrometer) in which pEC50 were 5.56-0.32 and 5.55+/-0.21, 5.99+/- 1.16 and 5.57+/-0.34, respectively. These probes except higenamine also relaxed KCl (65.4 mM)-contracted aorta. In isolated rat atria, all THIs and dobutamine increased heart rate and contractile force. In the presence of propranolol, the concentration response curves of YS 49 and YS 51 shifted to the right and resulted in pA2 values of 8.07+/-0.84 and 7.93+/-0.11, respectively. The slope of each compound was not deviated from unity, indicating that these chemicals are highly competitive at the cardiac beta-adrenoceptors. YS 49 YS 51, and higenamine showed alpha-adrenoceptor affinity in rat brain, in which the dissociation constant (Ki) was 2.75, 2.81, and 1.02 micrometer, respectively. It is concluded, therefore, that THI alkaloids have weak affinity to alpha1-adrenoceptors in rat aorta and brain, respectively, while these probes show relatively high affinity for cardiac beta-adrenoceptors. Thus, these chemicals may be useful in the treatment of congestive heart failure.

Pharmacological characterization of synthetic tetrahydroisoquinoline alkaloids, YS 51 and YS 55, on the cardiovascular system

Tetrahydroisoquinoline (THI) alkaloids can be considered as cyclized derivatives of simple phenylethylamines, and many of them, especially with 6,7-disubstitution, demonstrate relatively high affinity for catecholamines. Two -OH groups at 6 and 7 positions are supposed to be essential to exert beta-receptor activities. However, it is not clear whether -OH at 6,7 substitution of THIs also shows alpha-adrenoceptor activities. In the present study, we investigated whether -OH or -OCH3 substitutions of 6,7 position of THIs differently affect the alpha1-adrenoceptor affinity. We synthesized two 1-naphthylmethyl THI alkaloids, 1-beta-naphthylmethyl-6,7-dihydroxy-1,2,3,4-tetrahydroisoquinoline HBr (YS 51) and 1-beta-naphthylmethyl-6, 7-dimethoxy-1,2,3,4-tetrahydroisoquinoline HCl (YS 55), and their pharmacological actions on alpha1-adrenoceptor were compared. YS 51 and YS 55, concentration-dependently relaxed endothelium-denuded rat thoracic aorta precontracted with phenylephrine (PE, 0.1 micrometer) in which pEC50 were 5.89+0.21 and 5.93+ 0.19, respectively. Propranolol (30 nM) did not affect the relaxation-response curves to YS 51 and YS 55. Concentration-response curves to PE were shifted to right by the pretreatment with YS 51 or YS 55. The pA2 values of YS 51 and YS 55 showed 6.05 + 0.24 and 5.88 + 0.16, respectively. Both probes relaxed KCl (65.4 mM)-contacted aorta and inhibited CaCl2-induced contraction of PE-stimulated endothelium-denuded rat thoracic aorta in Ca2+-free solutions. In isolated guinea pig papillary muscle, 1 and 10 micrometer YS 51 increased contractile force about 4- and 8- fold over the control, respectively, along with the concentration-dependent increment of cytosolic Ca2+ ions. While, 10 micrometer YS 55 reduced the contractile force about 50 % over the control and lowered the cytosolic Ca2+ level, in rat brain homogenates, YS 51 and YS 55 displaced (3H)prazosin binding competitively with Ki 0.15 and 0.12 micrometer, respectively. However, both probes were ineffective on (3H)nitrendipine binding. Therefore, it is concluded that two synthetic naphthylmethyl-THI alkaloids have considerable affinity to alpha1-adrenenoceptors in rat aorta and brain.

HIV-1 p24 Antigen Detection by Immune Complex Dissociation and Neutralization in HIV-1 Infected Persons in Korea / 감염

BACKGROUND: HIV-1 p24 antigen assay is useful for the detection of circulating viruses, and infection prior to seroconversion. However, circulating HIV-1 p24 antigen may be complexed with HIV antibody and prevent it from being detected by antigen capture assay. To detect HIV-1 p24 antigen in the specimen, it is necessary to dissociate immune complexes and confirm the presence of HIV-1 p24 antigen after the neutralization with the specific antibody. METHODS: We tested 32 sera from HIV-1 infected persons who were diagnosed from 1987 tO1996 in Korea for HIV-1 p24 antigen by enzyme linked immunosorbent assay (ELISA.) with or without the dissociation of immune complexes. And we confirmed the antigen assay results by the neutralization with HIV-1 specific antibody as a confirmatory test. We also calculated the concentration of HIV-1 p24 antigen in each specimen. RESULTS: Eleven of 32 sera tested were initially positive for HIV-1 p24 antigen. After the dissociation of immune complexes for 29 sera except two of which signal/cutoff ratios were higher than 7.0 and except one which was not enough for the assay,13 were shown to be positive for HIV-1 p24 antigen and their signal/cutoff ratios were increased by 10 times. Five of antigen negative cases were turned to be positive after the immune complex dissociation. After neutralization with HIV-1specific p24 antibody for sera of 13 which were positive for HIV-1 p24 antigen with or without the immune complex dissociation, all were shown to be neutralized. We have observed more than 90% neutralization reduction for 12 sera and more than 50% less than 90% for one. The average concentration of HIV-1 p24 antigen was8.76pg/ml by antigen assay and was increased to 76.81~g/m~ after immune complex dissociation. CONCLUSION: We concluded that the sensitivity and the specificity of HIV-1 p24 antigen assay could be increased by dissociation of the immune complexes and neutralization with the specific antibody.