• 한국응용약물학회:학술대회논문집
• /
• 한국응용약물학회 1994년도 춘계학술대회 and 제3회 신약개발 연구발표회
• /
• pp.192-192
• /
• 1994

본 논문에서는 cytochrome P450 LA1 유전자의 5'-upsteam 조절부위의 클로닝을 실시하였다. pUC19 vector에 연결시킨 3.4 Kb 크기의 Pstl DNA조각을 Sst1, Nco1 제한 효소로 자른 뒤, Exonuclease III 를 처리하여 약 200bp 씩의 차이를 갖는 여러 크기의 plasmid들을 얻었다. 이 plasmid 의 핵산서열을 알아보기 위해 dideoxy nucletide를 이용한 sequencing방법으로 그 핵산서열의 결정 실험을 시도하였다. 또한, 다환상 방향족 탄화수소 화합물에 반응성을 갖는 C57BL/6N 생쥐와 반응성을 갖지않는 DBA/2N 생쥐에 있어 phase II 대사 효소인UDP-glucuronosyltransferase 효소활성에 대한 3-methylcholanthrene의 영향을 알아보기 위해 C57BL/6N 생쥐와 DBA/2N 생쥐에 각각 다른 농도의 3-methylcholanthrene을 처리하거나 각기 다른 시간에 3-methylcholanthrene를 처리하였다. 그 결과 UDP-glucuronosyl-transferase의 mRNA가 3-methylcholanthrene양의 증가에 따라, 처치시간이 길어짐에 따라 증가되어지며 그 mRNA위 크기는 약 2.2Kb 정도임을 알았다. 이로부터 UDP-ghucuronosyltransferase 또한 cytochrome P45O와 함께 다환상 방향족 탄화수소 화합물 조절인자를 통한 조절을 받을 것이며 phase I phase II 약물 대사 효소가 조절상 밀접한 관련을 가짐을 예측할 수 있었다.

• 한국식품영양과학회지
• /
• 제37권5호
• /
• pp.612-617
• /
• 2008

우리나라 남해안 연안에 서식하는 복섬(Takifugu niphobles) 간장의 TTX유도체들을 활성탄 칼럼을 이용하여 부분 정제하고, Hydrophilic Interaction Liquid Chromatography(HILIC)를 사용한 LC/MS(SIM mode)로 분석하였으며, pH와 가열 조건에 따른 독성 변화를 조사하고 복섬 fillet으로 복국 조리시 독성의 분포를 조사하였다. 복섬 간장의 독성분은 LC/MS에 의하여 7개의 성분이 분석되었으며, 각 성분의 함량과 조성은 5,6,11-trideoxyTTX(34.0%, 1,029.6nmol/g), 6,11-dideoxyTTX(29.3%, 887.6 nmol/g), TTX (22.1%, 667.8 nmol/g), 4,9-anhydroTTX(11.2%, 339.3nmol/g), 11-deoxyTTX＋5-deoxyTTX(2.6%, 78.6 nmol/g), 4-epiTTX(0.8%, 23.6 nmol/g), 5,6,11-trideoxyTTX(34.0%), 6,11-dideoxyTTX(29.3%), TTX(22.1%), 4,9-anhydroTTX (11.2%), 4-epiTTX(0.8%)이었다. 복섬 간장 추출물 희석액의 독성은 pH에 따라 크게 변하여 pH 3에서 8.4 MU/mL로 최고의 독성을 나타내었고, 알칼리로 갈수록 독성이 감소하여 pH 10에서는 pH 3의 1/7(1.4 MU/mL)을 나타내었다. 각각의 pH(pH 5, 7, 9)에서 $80^{\circ}C,\;100^{\circ}C,\;115^{\circ}C$를 유지하며 가열 시 온도는 높을수록, 시간은 길수록 독성은 감소하였다. 특히, 산성이나 중성에서는 독성이 완만하게 감소하는 경향을 보였으나, 알칼리 영역인 pH 9에서는 가열 10분 후에 $80^{\circ}C$의 경우라도 최초 독성(79 MU/mL)이 1/2 이하로 급격히 감소하였으며, $115^{\circ}C$에서는 완전 소멸하였다. 복섬 개체별 fillet 중의 총 독량은 $43.2{\sim}106.7$ MU로 개체에 따라서 2.5배까지 독량의 차이를 나타내었으며, 복섬 가식부에 3배량의 물을 가하여 10분간 끓일 경우 총 독량의 $64{\sim}78%$는 국물 중으로 용출되어 나와 육에 남아있는 것보다 독성이 강하였다.

• Bulletin of the Korean Chemical Society
• /
• 제25권2호
• /
• pp.221-225
• /
• 2004

A series of 2',3'-dideoxy-3'-fluoro-D-apiosyl nucleosides 15, 16, 17 and 18 were synthesized enantiomerically with L-Gulonic- ${\gamma}$-lactone as the starting material. The reduction of butenolide 1 with DIBAL-H followed by the Luche procedure afforded the allylic alcohol 2. Ozonolysis and the reduction of compound 4 induced the cyclized lactol, which was acetylated to give the acetate 7. Condensation of the acetate 7 with silylated pyrimidine ($N^4$-benzoyl cytosine) and a purine base (6-chloropurine) under Vorbruggen conditions and deblocking afforded a series of fluorinated apiosyl nucleosides.

• 미생물학회지
• /
• 제24권3호
• /
• pp.236-242
• /
• 1986

The nucleotide sequence of the genetic control site of Bacillus subtilis gene for $(1-4)-{\beta}-D-glucan$ endoglucanase (cellulase) was determined according to the procedures of the dideoxy chain termination method(Sanger et. al., 1977). The deduced amino acid sequence of this enzyme has a hydrophobic signal peptide at the $NH_2$ terminus similar to those found in fifteen other extracellualr enzymes from Bacillus species. This is followed by a sequence resembling the Bacillus ribosome binding site 14 nucleotide before the first codon of the gene. The presumptive promoter sequence was located 92 base pairs upstream fromthe initiation codon. The homology region in signal sequences was striking when comparing all the signal sequences of sixteen extracellular enzymes from Bacillus species so far compiled.

• 대한수의학회지
• /
• 제32권4호
• /
• pp.555-567
• /
• 1992

In this study gene encoding structural proteins of a CPV isolate was cloned and sequenced to elucidate the molecular genetical properties of the canine parvoviruses isolated from the field. Six recombinant plasmids of pEP3, p1471, p2070, pEP069, pEP338 and p14711p were constructed from the map positions 22 to 98 of RF DNA to clone the VP1 and VP2 genes of CPV-V20. Sequentialy the gene comprising 3780 nucleotides were sequenced by dideoxy chain termination method. When nucleotide sequence of gene encoding the structural proteins of CPV-V20 was compared with those of other strains, CPV-N, CPV-d and CPV-780929 published previously, DNA, homologies to CPV-V20 were 99.87% with CPV-N, 99.73% with CPV-d, 96.85% with CPV-780929 and 98.4% with FPLV-Carl, respectively. The DNA sequence data of CPV-V20 showed seven point mutations and also deletion of 135 nucleotides from the nucleotide position 4745 to 4879 located in the 3'-noncoding region of CPV-N.

• 한국양식학회지
• /
• 제8권3호
• /
• pp.209-220
• /
• 1995

미꾸라지의 간으로부터 핵을 분리하여, 저농도 염추출 및 제한효소 처리로 핵기질(nuclear matrix)을 분리하였다. 분리된 핵기질을 Proteinase K로 분해한 후, phenol-chloroform 추출로 크기가 약 0.3kb-15kb의 분포를 나타내는 핵기질 부착 DNA (nuclear matrix attachment regions : MARs)를 얻었다. 효모 URA 3 유전자를 가진 2.13 kb Eco47 III 단편을 제한효소 Ssp I 으로 절단된 pUC19 플라즈미드 벡타에 결합시켜, ARS (autonomously replication sequence) 클로닝을 위한 pURY19 플라즈미드 벡타를 만들었다. 이 pURY19 벡타는 Saccharomyces cerevisiae내에서 독립적으로 복제할 수 없기 때문에, 물고기의 효율적인 발현 벡타 개발을 위해, 이 system을 이용하여, S. cerevisiae내에서 독립적으로 복제 가능한 미꾸라지의 ARS를 클로닝하고자 하였다. 분리 된 MARs를 pURY19 벡타에 결합시 킨 다음, E. coli $DH5\alpha$에 형질전환시켜 $pURY19N_{l-62}$를 얻었다. MAR Libraries $(pURY19N_{1-62})$를 각각 $Ura^-\;S.\;cerevisiae$에 형질전환시켜, S. cerevisiae내에서 독립적으로 복제 가능한 M. mizolepis 유래의 복제원점들 (ARSs)을 분리하여, Sanger's dideoxy-chain termination method로 염기서열을 분석하였다. 염기서열 분석결과 모든 clones들은 AT-rich하였으며, 특히 $pURY19N_6$에는 ARS concensus sequence, Topoisomerase II consensus, near A-box, 그리고 T-box들이 존재하였다.

• 대한수의학회지
• /
• 제38권1호
• /
• pp.53-63
• /
• 1998

Total of 1085 swine sera (1996-1997) from nation-wide were tested for the presence of antibodies to influenza A virus. Fifty nine percent of the tested sera showed seropositive by HI test. Positive sera consisted of 24--- of H3, 15--- of H1, and 20--- of the sample had both antibodies, respectively. Sera collected from various region represented 7~27--- seropositivity to H1N1, 15~25--- to H3N2, respectively. Swine influenza field isolate from nasal swab was characterized antigenically and genetically to elucidate its relatedness with other known strains of influenza A virus. The study was focused on the HA gene which is related to pathogenecity and antigenic variability of the influenza virus. By RT-PCR using influenza A/H1N1 specific primers, influenza virus H1N1 specific DNA fragment was amplified from A/Swine/Iowa/15/30(H1N1), US field isolate but not in H3N2 strain. PCR products were sequenced by dideoxy chain termination method to determine nucleotide homology with other strains of influenza A virus. The US field isolate and A/Swine/Indiana/1726/88 strain had 97--- of nucleotide homology and 98--- of amino acid homology. Based on the results obtained from this experiment, the field isolate was genetically related to A/Swine/Indiana/1726/88 and had higher homology with A/Swine/Indiana/1726/88 than with classical swine influenza virus, A/Swine/Iowa/15/30. The field isolate had no amino acid changes at the antigenic site compare to that of the A/Swine/Indiana/1726/88. The proteolytic enzyme cleavage site between HA1 and HA2 had no alteration and the amino acid arginine was intact. There is no evidence has been found that the field isolate has genetic shift or genetic drift which might altered antigenic determinant.

• Advances in Traditional Medicine
• /
• 제2권2호
• /
• pp.87-93
• /
• 2002

The present study was aimed to examine the role of endothelium in the relaxant effect of hawthorn fruit extract of Crataegus pinnatifida in four different types of rat arteries, posterior cerebral communicating artery, right descending coronary artery, common carotid artery, and aorta. In $9,11-dideoxy-11{\alpha}$, $9{\alpha}-epoxy-methanoprostaglandin$ $F_{2{\alpha}}$ (U46619)-preconstricted arterial rings except for aorta, the extract produced endothelium-independent relaxations with similar potency. This relaxation was unaffected by pretreatment with $100\;{\mu}M\;N^G-nitro-L-arginine$ methylester (L-NAME, the nitric oxide synthase inhibitor), $3\;{\mu}M$ 1H-[l,2,4]oxadiazolo$[4,2-{\alpha}]$quinoxalin-1-one (ODQ, the guanylate cyclase inhibitor), or $10\;{\mu}M$ indomethacin (the cyclooxygenase inhibitor). Putative $K^+$ channel blockers (charybdotoxin plus apamin or glibenclamide) did not affect the extract-induced relaxation in cerebral or coronary artery rings. In contrast, in rat aortic rings the extract produced significantly smaller relaxant response in endothelium-denuded rings than that in endothelium-intact rings. Pretreatment with L-NAME or ODQ abolished the extractinduced endothelium-dependent aortic relaxation, whilst indomethacin $(3\;{\mu}M)$ had no effect. The present results indicate that hawthorn fruit extract possesses a vasorelaxing effect in cerebral, coronary and carotid arteries and this effect is independent of the presence of a functional endothelium. However, the extract-induced endothelium-dependent relaxation in rat aorta was mediated through endothelial nitric oxide and cyclic GMP-dependent mechanisms, suggesting that active components in the extract may act on endothelium to stimulate release of nitric oxide in large conduit arteries of the rats.

• Archives of Pharmacal Research
• /
• 제29권5호
• /
• pp.375-383
• /
• 2006

The anti platelet effects of a novel guanidine derivative, KR-32570 ([5-(2-methoxy-5-chlorophenyl) furan-2-ylcarbonyl]guanidine), were investigated with an emphasis on the mechanisms underlying its inhibition of collagen-induced platelet aggregation. KR-32570 significantly inhibited the aggregation of washed rabbit platelets induced by collagen $(10{\mu}g/mL)$, thrombin (0.05 U/mL), arachidonic acid $(100{\mu}M)$, a thromboxane (TX) $A_2$ mimetic agent U46619 (9,11-dideoxy-9,11-methanoepoxy-prostaglandin $F_2,\;1{\mu}M$) and a $Ca^{2+}$ ATPase inhibitor thapsigargin $(0.5{\mu}M)$ ($IC_{50}$ values: $13.8{\pm}1.8,\;26.3{\pm}1.2,\;8.5{\pm}0.9,\;4.3{\pm}1.7\;and\;49.8{\pm}1.4{\mu}M$, respectively). KR-32570 inhibited the collagen-induced liberation of $[^3H]$arachidonic acid from the platelets in a concentration dependent manner with complete inhibition being observed at $50{\mu}M$. The $TXA_2$ synthase assay showed that KR-32570 also inhibited the conversion of the substrate $PGH_2$ to $TXB_2$ at all concentrations. Furthermore, KR-32570 significantly inhibited the $[Ca^{2+}]_i$ mobilization induced by collagen at $50{\mu}M$, which is the concentration that completely inhibits platelet aggregation. KR-32570 also decreased the level of collagen $(10{\mu}g/mL)$induced secretion of serotonin from the dense-granule contents of platelets, and inhibited the NHE-1-mediated rabbit platelet swelling induced by intracellular acidification. These results suggest that the antiplatelet activity of KR-32570 against collagen-induced platelet aggregation is mediated mainly by inhibiting the release of arachidonic acid, $TXA_2$ synthase, the mobilization of cytosolic $Ca^{2+}$ and NHE-1.

• International Journal of Industrial Entomology and Biomaterials
• /
• 제27권2호
• /
• pp.237-242
• /
• 2013

We investigated the 1-deoxynojirimycin (1-DNJ) content of extracts from silkworm larvae at each developmental stage within three silkworm varieties. We also compared the content of the following polyhydroxylated alkaloids in the silkworm extracts: 1-DNJ, fagomine, and 1,4-dideoxy-1,4-imino-d-arabinitol (DAB). In addition, we evaluated the glucose-lowering effects of silkworm extract powder in db/db mice. The 1-DNJ content was the highest in Yeonnokjam $5^{th}$ instar $3^{rd}$ d larvae and Hansaengjam $5^{th}$ instar $3^{rd}$ d larvae, which contained 18.4 mg/100 g dry weight and 18.3 mg/100 g dry weight, respectively. The $5^{th}$ instar $3^{rd}$ d larvae exhibited a higher content of 1-DNJ than that of $5^{th}$ instar $5^{th}$ d larvae among all varieties. The glucose-lowering effects of silkworm extracts and Yeonnokjam powder were tested on db/db mice, and the blood glucose levels were found to decrease significantly in the YR70 group. Silkworm extracts (180 mg/kg, 90 mg/kg, 45 mg/kg, and 22.5 mg/kg) and acarbose (50 mg/kg) were administered orally for 4 wk. Changes in water intake were not statistically significant between control and silkworm extract-treated groups. Compared to the control group, blood glucose levels in the silkworm extract powder-treated group decreased in the 22.5 mg/kg/d group after being administered for 4 wk. This decrease was statistically significant. Furthermore, biochemical changes in the AST(Aspartate aminotransferase), ALT(Alanine aminotransferase), TCHO(Total Cholesterol), TG(Triglyceride), LDL(Low density lipoprotein), and HDL(High density lipoprotein) levels in blood were not observed. However, statistically significant decreases in blood GLU in the 22.5 mg/kg/d group compared to that of the control group occurred. In addition, the epididymal fat weight of the silkworm extract powder-treated group decreased significantly in both the 22.5 mg/kg/d group and 180 mg/kg/d group compared to that of the control group, but there were no statistically significant changes in perirenal fat weight. These results demonstrate that silkworm extracts inhibit changes in blood glucose levels in model diabetic mice.