The effect of an ethanolic extract of the plant Trianthema portulacastrum L. on the $CCI_4$-induced chronic hepatocellular damage of Swiss albino mice has been investigated. The normal mice received olive oil (0.2 ml/mouse) for five weeks. The $CCI_4$ control mice, on the other hand, received $CCI_4$ (0.05 ml/mouse) in olive oil for five weeks. The extract was administered at the dose of 100 mg/kg or 150 mg/kg for five weeks by gastric intubation in addition to $CCI_4$ treatment. The $CCI_4$ administraction alone caused hepatocellular necrosis, severe anemia, leucopaenia, lymphocytopaenia, neutrophilia, eosinophilia and haemoglobinaemia along with the alterations of plasma albumin and globulin. The administration of plant extract (at 100 or 150 mg/kg) restored the $CCI_4$-induced alterations of the haematological parameters to the normal level. The extract of T. portulacastrum elicited a marked protection against $CCI_4$-induced hepatotoxicity as indicated by the several haematological parameters, related indices of formed elements, and different fractions of plasma protein. We also observed the dose-dependent antihepatotoxic effect of the extraction on these mice. The 150 mg/kg of extract was found to be more effective in normalizing the toxic effects of $CCI_4$ on the above parameters of mice. These results suggest that the hepatoprotective effect of T. poltulacastrum could be caused by its critical involvement in modulating several factors associated with erythropoiesis, and the boosting of general immunity of the host.

A sensitive enzyme immunoassay for serum TSH has been developed utilizing the tight binding between biotin and avidin, and three layered protein polystyrene beads as solid phase. To increase binding capacity of TSH and sensitivity of the assay, the polystyrene beads were coated sequentially with mouse immunoglobulin as first layer, rabbit antimouse immunoglobulin as second layer and monoclonal anti-TSH as third layer. A serum sample was incubated simultaneously with a monoclonal anti-TSH immobilized polystyrene beads and a second monoclonal anti-TSH covalently attached to biotin. After washing, the antibody bound serum TSH-anti-TSH-biotin complex is reacted with horseradish peroxidase (HRP)-labeled avidin. Following second wash, the bound HRP activity was measured calorimetrically. Reproducible results were obtained within 4 hours for serum TSH in the range between $0{\mu}\textrm{IU}$ml and ${50}{\mu}\textrm{IU}$ml with detection limit of $0.1{\mu}\textrm{IU}$ per test.

The effect of thyroid hormones on the hepatic xanthine oxidase activity was studied in rats after the intraperitoneal injections of comthyroid (triiodotyronine:thyroxine=1:4) at 0.3 mg/kg for 3 consecutive days. The aim of this study was to understand the precise mechanism of hyperthyroidism induced by oxidative stress. The concentration of lipid peroxides determined indirectly by the measurement of thiobarbituric acid reactants was increased in comthyroid treated rats. The hepatic glutathione content was decreased in comthyroid injected rat compared to the euthyroid state. It was also observed that the increment of xanthine oxidase activity has a profound role in oxygen radicals generation system in comthyroid treated rat. These findings suggest that the enhanced xanthine oxidase activity and depleting glutathione content in comthyroid treated rats result in pathophysiological oxidative stress including an increment of hepatic lipid peroxidation.

This study was designed to determine biochemical and pharmacological properties of a newly synthesized benzimidazole derivative, 2-amino-4, 5-dihydropyrido [1, 2-a] thiazolo [5, 4-g] benzimidazole (YJA20379-5) in vitro and in vivo. In the leaky membrane vesicles of pig gastric mucosa, YJA20379-5 inhibited the $K^+$-stimulated $H^+$, $K^+$-ATPase activity in a concentration- and time-dependent manner, with $IC_{50}$ values being $43{\mu}\textrm{M}$ and $43{\mu}\textrm{M}$ at pH 6.4 and 7.4, respectively. YJA20379-5, given intraduodenally, had a potent inhibitory effect on the gastric acid secretion in pylorus-ligated rats. The $ED_{50}$ value for acid secretion was 15.4 mg/kg. YJA20379-5, administered orally, also suppressed gastric damages induced by water-immersion stress, indomethacin and ethanol, and duodenal damage induced by mepirizole in rats; the $ED_{50}$ values were 17.6, 4.7, 3.0 and 18.7 mg/kg, respectively. Furthermore, repeated oral administration of YJA20379-5 accelerated the spontaneous healing of acetic acid-induced gastric ulcers in rats. It is concluded that the a-ntisecretory activity of YJA20379-5 appears to be associated with inhibition of $H^+$, $K^+$-ATPase, while its antigastric and antiduodenal lesion activities are primarily related to the antisecretory effect.

To prolong the biological half-life of streptokinase, a thrombolytic agent, streptokinase-bearing liposome with and distearolyphosphatidyl ethanolamine-N-poly (ethylene glycol) 2000 (DSPEPEG 2000) was prepared and evaluated. Streptokinase-bearing liposomes composed of distearolyphosphatidylcholine (DSPC), cholesterol and cholesterol-3-sulfate with DSPE-PEG 2000 was prepared by the freeze-thawing method and administered via femoral vein to rats (15000 IU/kg). The activity of streptokinase in plasma was determined by the method based on the amidolytic activity of streptokinase-plasminogen complex. Pharmacokinetic parameters of streptokinase incorporated in liposomes were compared with those of streptokinase alone. The $T_{1/2}$ and $AUC_\infty$ streptokinase incorporated in DSPC-PEG liposome increased 16.3- and 6.1-fold, respectively, compared with those of streptokinase alone. Streptokinase-bearing long-circulating liposome could increase the circulation time of streptokinase in blood and expect longer thrombolytic activity compared with streptokinase alone.

In this study, we developed immunoassay methods for the more convenient and effective detection of rat tracheal mucin and the results were compared with those of [$3^H$]glucosamine based gel-filtratioh method. A monoclonal anti-rat tracheal mucin antibody, mAbRT03, which specifically recognizes rat tracheal mucins, was used throughout in this study. To induce mucin secretion, varying concentrations of ATP (0-2 mM) were applied to the primary rat tracheal surface epithelial (RTSE) cell culture which had been metabolically radiolabeled with [$3^H$]glucosamine and the secretion of mucin was analyzed both by the immunoassay and the gel-filtration chromatography methods. For the immunoassay, the following two procedures were employed. 1) Simple ELISA; the culture spent media were directly coated onto the assay plate and the immunoreactivity with mAbRT03 was assessed from the standard curve generated with the purified rat mucin. 2) Inhibition ELISA; A known amount of the purified rat mucin was coated onto the assay plate and then ATP-stimulated culture spent media were added to inhibit the immunorelitivity with mAbRT03. The contents of mucin in the sample were calculated from the standard inhibition curve which was generated with the purified rat mucin. The assay results obtained from the immunoassays were identical with those from the gel-filtration methods. The present result indicates that ELISA can be substituted for the laborious, time-consuming gel-filtration assay in studying the regulation of airway mucin release from cultured airway epithelial cells.

Angiogenic activity of Aloe vera gel was investigated by in vitro assay. We obtained the most active fraction from dichloromethane extract of Aloe vera gel by partitioning between hexane and 90% aqueous methanol. The most active fraction (F3) increased the proliferation of calf pulmonary artery endothelial (CPAE) cells. In addition, F3 fraction induced CPAE cells to invade type I collagen gel and form capillary-like tube through in vitro angiogenesis assay, and increased the invasion of CPAE cells into matrigel through in vitro invasion assay. Furthermore, the effect on the MRNA expression of proteolytic enzymes which are key participants in the regulation of extracellular matrix degradation was investigated by northern blot analysis. F3 fraction enhanced mRNA expression of urokinase-type plasminogen activator (u-PA), matrix metalloproteinase-2 (MMP-2), and membrane-type MMP (MT-MMP) in CPAE cells whereas the expression of plasminogen activator inhibitory (PAl-1) mRNA was not changed.

The present study investigated the antitumor activity of the aqueous-alcoholic extracts from unripe cotton balls of Gossypium indicum. An Exposure of murine B16 melanoma and L1210 lymphoma cells to the extracts resulted in their severe deaths in time- and concentration-dependent manners. Of the extracts, hydrophilic fractions were most efficacious for the antitumor activity and found to contain certain amounts of catechin and its derivatives. The hydrophilic extract fraction C36B2-8 had approximately 10 times more cytotoxic effects on B12 and L1210 cells than on isolated murine thymocytes. High concentrations (>150 $\mu$g/ml) of C 36B3-8 mainly induced necrotic cell death. At low concentrations (<100 $\mu$g/ml), however, C 36B3-8 induced not only necrosis but also apoptosis of the two tumor cell lines, which was proved by the TUNEL staining and DNA fragmentation techniques. The data indicate that certain ingredients of the cotton ball extract of G. indicum have an antitumor activity.

In order to determine the structure-activity relationships for antioxidative effects of gagaminine, a steroidal alkaloid isolated from the roots of Cynanchum wilfordi (Asclepiadaceae), two derivatives identified as sarcostin and penupogenin were prepared from gagaminine by hydrolysis and reduction. These compounds were evaluated for the inhibitory effects on the aidehyde oxidase activity and on lipid perbxidation in vitro. Furthermore, their effects were compared with those of gagaminine and the related compounds, cinnamic acid and nicotinic acid. The results of this study prove that the cinnamoyl group in the structure of gagaminine is critical in inhibition of the aldehyde oxidase activity while the nicotinoyl group may be necessary for anti-lipid peroxidation of the compound.

The microdilution assay recommended by NCCLS (National Committee for Clinical Laboratory Standards) is one of the standardized methods of antibiotic susceptibility test. This method has been widely used clinically to obtain MIC values of antibiotics on pathogenic microorganisms. It is more convenient, rapid and simple to test many samples than other test methods such as agar diffusion assay and broth macrodilution assay. The screening of antimicrobial agents from microbial extracts is too laborious in its process. Therefore, a number of screening methods having more simple procedure have been developed. In our laboratory, we applied microdilution assay for screening the antimicrobial agents. This assay showed dose-response results and was more sensitive than disc diffusion assay in our system. We tested 200 samples of microbial extracts originated from 100 microbial strains and selected several samples as potential candidates. In this report, we show that the microdilution assay is more convenient method in screeing of antibiotic susceptibility than those previously reported.

The abuse of zipeprol, an antitussive agent, was found to be most prevalent among young people in Korea. Because abusers take large doses of this drug for its hallucinogenic effects, fatalities from zipeprol overdose abuse have been on the rise since 1991. Since 1991, a total of 69 zipeprol-related deaths have occurred throughout the nation. A demographic study shows that in ninety six percent of cases involving zipeprol alone, the victims were in their teens and twenties. The mate/female ratio in zipeprol related death was 3.5:1. Most of these zipeprol-associated deaths occurred in the larger cities of Seoul and Inchon. The blood concentration of zipeprol ranged from 0.8 to $38.3{\mu}g/mL $in single drug involved deaths, while zipeprol varied from to 35.3 $0.1{\mu}g/mL $in zipeprol and dextromethorphan victims.

Cop protein has been overexpressed in Escherichia coli using a T7 RNA polymerase system. Purification to apparent homogeneity was achieved by the sequential chromatography on ion exchange, affinity chromatography, and reverse phase high performance liquid chromatography system. The molecular weight of the purified Cop was estimated as 6.1 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). But the molecular mass of the native state Cop was shown to be 19 kDa by an analytical high performance size exclusion chromatography, suggesting a trimer-like structure in 50 mM Tris-HCI buffer (pH 7.5) containing 100 mM NaCl. Cop protein Was calculated to contain $39.1% {\alpha}-helix, 16.8% {\beta}-sheet$, 17.4% turn, and 26.8% random structure. The DNA binding property of Cop protein expressed in E. coli Was preserved during the expression and purification process. The isoelectric point of Cop was determined to be 9.0. The results of amino acid composition analysis and N-terminal amino acid sequencing of Cop showed that it has the same amino acid composition and N-terminal amino acid sequence as those deduced from its DNA sequence analysis, except for the partial removal of N-terminal methionine residue by methionyl-aminopeptidase in E. coli.

Retinoids are applied to not only cancer prevention but also cancer chemotherapy by stimulating differentiation of cells. We studied differentiation inducing effect of all-trans retinoic acid (ATRA) by studying proportion of high dense fractions of stem-like cells and the size of S phase fraction in cell cycle. From mammary organoids obtained from 7- to 8-week old F344 female rat mammary gland, we cultured rat mammary epithelial cells (RMEC) and treated physiological doses of $10^{-6}$, $10^{-7}$, and $10^{-8}$ M ATRA from the first day and then cultured for 4, 7, and 14 days. After that, immunostaining was performed using peanut agglutinin (PNA) and anti-Thy-1.1 monoclonal antibody (Thy-1.1) that can be used as markers of differentiation. We separated four different cell subpopulations by flow cytometry: cells negative to both reagents (B-), PNA-positive cells (PNA+), Thy-1.1-positive cells (Thy-1.1+), and cells positive to both reagents (B+). We observed continuous decreases of high dense fractions of stem-like cells (PNA+ subpopulations) for 14 days and as much decreases as high doses of ATRA, which were thought to be proportional to doses of ATRA. We labeled RMEC with bromodeoxyuridine and investigated cell cycle fractions that went through S phase. We observed a tendency of decrease of S phase fraction with time in culture, which, is thought to be related to continuous decreases of PNA+ subpopulations and inhibitory role of ATRA on cell cycle. These results suggest that physiological doses of ATRA could stimulate differentiation of RMEC and convert stem-like RMEC to differentiated cells in SFM for a relatively long period of 14 days.

Human cytochrome P450 1A2 is one of the major cytochrome P450s in human liver. It is known to be capable of activating a number of carcinogens such as arylamines and heterocyclic amines. In order to develop the new bacterial mutagenicity test system with human P450, a full length of human P450 1A2 cDNA inserted into pCW bacterial expression vector was introduced to Escherichia coli WP2 uvrA strain which is a well-known E. coli strain for bacterial reverse mutagenicity assay. Expressed human P450 1A2 showed typical P450 hemoprotein spectra. Maximum expression was achieved at 48 hrs after incubating at $30^{\circ}C$ in terrific broth containing ampicillin, IPTG and other supplements. High level expression of P450 1A2 in E. coli WP2 uvrA membranes was determined in SDS-PAGE. The well-known mutagens 2-aminoanthracene and MElQ increased the revertant colonies of E. coli WP2 uvrA expressing human P450 1A2 without an exogenous rat hepatic post-mitochondrial supernatant (S9 fraction) in a dose-dependent manner. The results show that the functional expression of human P450 in bacterial mutagenicity tester strain will provide a useful tool for studying the mechanism of the mutagenesis and carcinogenesis of new drugs and environmental chemicals.

DW-116 is a new fluoroquinolone antimicrobial agent with a broad spectrum. In order to elucidate the resistance mechanism to DW-116 in Acinetobacter spp. bacteria, total chromosomal DNA was isolated from 10 strains of Acinetobacter spp. resistant to DW-116. Quinolone resistance determinant region (QRDR) of DNA gyrase gene was amplified by PCR. The 345 bp nucleotide fragment yielded was inserted into pKF 3 which was used as the vector. Comparisons of the DNA sequences of 8 strains with that of the wild type strain revealed a Ser-83 to Leu mutation in mutants and all ten strains contained one silent mutation$(T{\rightarrow}G)$in QRDR. From Acinetobacter MB4-8 strain, DNA gyrase was isolated and purified, through novobiocin-sepharose, heparin-sepharose affinity column chromatography. The enzyme was composed of two subunits and the molecular mass of subunits A and B were 75.6 and 51.9 kDa, respectively. The supercoiling activity of the reconstituted DNA gyrase composed of subunit A from Acinetobacter MB4-8 and subunit B from E. coli was not inhibited by $128{\mu}\textrm{g}$ml of ciprofloxacin. It might be said that one of the resistance mechanisms to DW-116 in Acinetohacter MB4-8 was subunit A alteration of DNA gyrase.

Gastric smooth muscle of cats was used to investigate the involvement of protein kinase in vanadate-induced contraction. Vanadate caused a contraction of cat gastric smooth muscle in a dose-dependent manner. Vanadate-induced contraction was totally inhibited by 2 mM EGTA and 1.5 mM $LACI_3$ and significantly inhibited by $10\mu$M verapamil and $1\mu$M nifedipine, suggesting that vanadate-induced contraction is dependent on the extracellular $Ca^{2+}$ concentration, and the influx of extracellular $Ca^{2+}$ was mediated through voltage-dependent $Ca^{2+}$ channel. Both protein kinase C inhibitor and tyrosine kinase inhibitor significantly inhibited the vanadate-induced contraction and the combined inhibitory effect of two protein kinase inhibitors was greater than that of each one. But calmodulin antagonists did not have any influence on the vanadate-induced contraction. On the other hand, both forskolin ($1\mu$M) and sodium nitroprusside ($1\mu$M) significantly inhibited vanadate-induced contraction. Therefore, these results suggest that both protein kinase C and tyrosino kinase are involved in the vanadate-induced contraction which required the influx of extracellular $Ca^{2+}$ in cat gastric smooth muscle, and that the contractile mechanism of vanadate may be different from that of agonist binding to its specific receptor.

To elucidate of role(s) of tTA as a repressor in the tTA-mediated gene repression system, we introduced mutations into the acidic domain of VP16 and examined the effects of such various mutations. In the transient repression experiment, a region containing 34 amino acids of the activation domain of VP16 (412-456) which interacts with TFIIB was found to be necessary and sufficient for the tTA-mediated repression of gene expression. However, in the experiment to investigate the fact that tTA-regulated repression is related to the activation function of VP16, we found that the repression abilities of tTA derivatives did not correlate exactly with their activation abilities. Therefore, we conclude that increased mass of VP16 in tTA might be also important for efficient repression in addition to functional domain of VP16.

Four derivatives of 6-oxo-3,4,4a,5-tetrahydro-3-hydroxy-2,2-dimethylnaphtho-1,2-pyran (1), known as bacterial, bacteriostatic, fungicidal, fungistatic agents, were synthesized to investigate the effect of substituents on the aromatic ring.

N-(tert-Butoxycarbonyl)-O-(dimethythiophosphono)-L-tyrosine (6) and N-(tert-butoxycarbonyl)-O-(dicyanoethylthiophosphono)-L-tyrosine (15) were prepared as intermediates for the synthesis of thiophosphotyrosine-containing peptides. The reactivity and suitability of two compounds for the solid phase or solution phase peptide synthesis utilizing t-Boc chemistry were examined.

$2^1$-Fluorohexopyranosyl nucleosides 1a and 1b which contained a bioisosteric double bond and a fluorine were synthesized in 12 steps, starting from D-galactose. During diethylaminosulfur trifluoride (DAST) fluorination, retention of stereochemistry was observed through the participation of methoxy or chloro group at the 6-posiition of the purine base. The final nucleosides 1a and 1b were found to be inactive against HIV-1 and HSV-1,2.

To overcome multidrug resistance (MDR) in cancer chemotherapy, we prepared various plant extracts and searched for a component which is effective for inhibition of MDR. MDR inhibition activity was determined by measuring cytotoxicity to MDR cells using multidrug resistant human fibrocarcinoma KB V20C, which is resistant to 20 nM vincristine and expresses high level of mdr1 gene. Of various plant extracts, the MeOH extract of the root of Aconitum pseudo-laeve var. erectum was found to have potent inhibitory activity on MDR. The bioassayguided fractionation of the MeOH extract of the plant led to the isolation of an alkaloid, lycaconitine, as an active principle. And the $IC_{50}$ of lycaconitne for KB V20C cells was $74\mu{g}$/ml.

Different bacteria wwere seperated from saliva and teeth of cariogenic patients and identified by a variety of morphological and biochemical tests. Extracts of green tea strongly inhibited Escherichia coli, Streptococcus salivarius and Streptococcus mutans. The antibacterial effect of green and black tea extracts were compared with those of amoxicillin, cephradine and eugenol.

Geraniol (1), olivetol (2), cannabinoids (3 and 4) and 5-fluorouracil (5) were tested for their growth inhibitory effects against human oral epitheloid carcinoma cell lines (KB) and NIH 3T3 fibrobalsts using two different 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) assay and sulforhodamine B protein (SRB) assay. Cannabigerol (3) exhibited the highest growth-inhibitory activity against the cancer cell lines.

Two new 4-hydroxybenzyl alcohol derivatives (1 and 2) were isolated from the methanol extract obtained from fresh tubers of Gastrodia elata together with 4-hydroxybenzyl methyl ether, 4-hydroxybenzyl alcoho, bis(4-hydroxyphenyl)methane, 4-hydroxybenzaldehyde, $\beta$-sitosterol and plamitic acid. 1 and 2 were identified as 3-O-($4^1$-hydroxybenzyl)-$\beta$-sitosterol and 4-[$4^1$-($4^{11}$-hydroxybenzyloxy)benzyloxy]benzyl methyl ether, respectively, according to the spectroscopic data.