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Sesquiterpenoids Bioconversion Analysis by Wood Rot Fungi

  • Lee, Su-Yeon;Ryu, Sun-Hwa;Choi, In-Gyu;Kim, Myungkil
    • 한국균학회소식:학술대회논문집
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    • 2016.05a
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    • pp.19-20
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    • 2016
  • Sesquiterpenoids are defined as $C_{15}$ compounds derived from farnesyl pyrophosphate (FPP), and their complex structures are found in the tissue of many diverse plants (Degenhardt et al. 2009). FPP's long chain length and additional double bond enables its conversion to a huge range of mono-, di-, and tri-cyclic structures. A number of cyclic sesquiterpenes with alcohol, aldehyde, and ketone derivatives have key biological and medicinal properties (Fraga 1999). Fungi, such as the wood-rotting Polyporus brumalis, are excellent sources of pharmaceutically interesting natural products such as sesquiterpenoids. In this study, we investigated the biosynthesis of P. brumalis sesquiterpenoids on modified medium. Fungal suspensions of 11 white rot species were inoculated in modified medium containing $C_6H_{12}O_6$, $C_4H_{12}N_2O_6$, $KH_2PO_4$, $MgSO_4$, and $CaCl_2$ for 20 days. Cultivation was stopped by solvent extraction via separation of the mycelium. The metabolites were identified as follows: propionic acid (1), mevalonic acid lactone (2), ${\beta}$-eudesmane (3), and ${\beta}$-eudesmol (4), respectively (Figure 1). The main peaks of ${\beta}$-eudesmane and ${\beta}$-eudesmol, which were indicative of sesquiterpene structures, were consistently detected for 5, 7, 12, and 15 days These results demonstrated the existence of terpene metabolism in the mycelium of P. brumalis. Polyporus spp. are known to generate flavor components such as methyl 2,4-dihydroxy-3,6-dimethyl benzoate; 2-hydroxy-4-methoxy-6-methyl benzoic acid; 3-hydroxy-5-methyl phenol; and 3-methoxy-2,5-dimethyl phenol in submerged cultures (Hoffmann and Esser 1978). Drimanes of sesquiterpenes were reported as metabolites from P. arcularius and shown to exhibit antimicrobial activity against Gram-positive bacteria such as Staphylococcus aureus (Fleck et al. 1996). The main metabolites of P. brumalis, ${\beta}$-Eudesmol and ${\beta}$-eudesmane, were categorized as eudesmane-type sesquiterpene structures. The eudesmane skeleton could be biosynthesized from FPP-derived IPP, and approximately 1,000 structures have been identified in plants as essential oils. The biosynthesis of eudesmol from P. brumalis may thus be an important tool for the production of useful natural compounds as presumed from its identified potent bioactivity in plants. Essential oils comprising eudesmane-type sesquiterpenoids have been previously and extensively researched (Wu et al. 2006). ${\beta}$-Eudesmol is a well-known and important eudesmane alcohol with an anticholinergic effect in the vascular endothelium (Tsuneki et al. 2005). Additionally, recent studies demonstrated that ${\beta}$-eudesmol acts as a channel blocker for nicotinic acetylcholine receptors at the neuromuscular junction, and it can inhibit angiogenesis in vitro and in vivo by blocking the mitogen-activated protein kinase (MAPK) signaling pathway (Seo et al. 2011). Variation of nutrients was conducted to determine an optimum condition for the biosynthesis of sesquiterpenes by P. brumalis. Genes encoding terpene synthases, which are crucial to the terpene synthesis pathway, generally respond to environmental factors such as pH, temperature, and available nutrients (Hoffmeister and Keller 2007, Yu and Keller 2005). Calvo et al. described the effect of major nutrients, carbon and nitrogen, on the synthesis of secondary metabolites (Calvo et al. 2002). P. brumalis did not prefer to synthesize sesquiterpenes under all growth conditions. Results of differences in metabolites observed in P. brumalis grown in PDB and modified medium highlighted the potential effect inorganic sources such as $C_4H_{12}N_2O_6$, $KH_2PO_4$, $MgSO_4$, and $CaCl_2$ on sesquiterpene synthesis. ${\beta}$-eudesmol was apparent during cultivation except for when P. brumalis was grown on $MgSO_4$-free medium. These results demonstrated that $MgSO_4$ can specifically control the biosynthesis of ${\beta}$-eudesmol. Magnesium has been reported as a cofactor that binds to sesquiterpene synthase (Agger et al. 2008). Specifically, the $Mg^{2+}$ ions bind to two conserved metal-binding motifs. These metal ions complex to the substrate pyrophosphate, thereby promoting the ionization of the leaving groups of FPP and resulting in the generation of a highly reactive allylic cation. Effect of magnesium source on the sesquiterpene biosynthesis was also identified via analysis of the concentration of total carbohydrates. Our current study offered further insight that fungal sesquiterpene biosynthesis can be controlled by nutrients. To profile the metabolites of P. brumalis, the cultures were extracted based on the growth curve. Despite metabolites produced during mycelia growth, there was difficulty in detecting significant changes in metabolite production, especially those at low concentrations. These compounds may be of interest in understanding their synthetic mechanisms in P. brumalis. The synthesis of terpene compounds began during the growth phase at day 9. Sesquiterpene synthesis occurred after growth was complete. At day 9, drimenol, farnesol, and mevalonic lactone (or mevalonic acid lactone) were identified. Mevalonic acid lactone is the precursor of the mevalonic pathway, and particularly, it is a precursor for a number of biologically important lipids, including cholesterol hormones (Buckley et al. 2002). Farnesol is the precursor of sesquiterpenoids. Drimenol compounds, bi-cyclic-sesquiterpene alcohols, can be synthesized from trans-trans farnesol via cyclization and rearrangement (Polovinka et al. 1994). They have also been identified in the basidiomycota Lentinus lepideus as secondary metabolites. After 12 days in the growth phase, ${\beta}$-elemene caryophyllene, ${\delta}$-cadiene, and eudesmane were detected with ${\beta}$-eudesmol. The data showed the synthesis of sesquiterpene hydrocarbons with bi-cyclic structures. These compounds can be synthesized from FPP by cyclization. Cyclic terpenoids are synthesized through the formation of a carbon skeleton from linear precursors by terpene cyclase, which is followed by chemical modification by oxidation, reduction, methylation, etc. Sesquiterpene cyclase is a key branch-point enzyme that catalyzes the complex intermolecular cyclization of the linear prenyl diphosphate into cyclic hydrocarbons (Toyomasu et al. 2007). After 20 days in stationary phase, the oxygenated structures eudesmol, elemol, and caryophyllene oxide were detected. Thus, after growth, sesquiterpenes were identified. Per these results, we showed that terpene metabolism in wood-rotting fungi occurs in the stationary phase. We also showed that such metabolism can be controlled by magnesium supplementation in the growth medium. In conclusion, we identified P. brumalis as a wood-rotting fungus that can produce sesquiterpenes. To mechanistically understand eudesmane-type sesquiterpene biosynthesis in P. brumalis, further research into the genes regulating the dynamics of such biosynthesis is warranted.

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Legal Study for the KSLV launching - Products & Third Party Liability - (KSLV발사에 따른 제작 및 제3자피해 책임에 대한 우주법적 소고)

  • Shin, Sung-Hwan
    • The Korean Journal of Air & Space Law and Policy
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    • v.21 no.1
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    • pp.169-189
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    • 2006
  • In 2007, KSLV(Korea Small Launching Vehicle) that we made at Goheung National Space Center is going to launch and promotes of our space exploration systematically and 'Space Exploration Promotion Act' was enter into force. 'Space Exploration Promotion Act' article 3, section 1, as is prescribing "Korean government keeps the space treaties contracted with other countries and international organizations and pursues after peaceful uses of outer space." The representative international treaties are Outer Space Treaty (1967) and Liability Convention (1972) etc. In Liability convention article 2, "A launching State shall be absolutely liable to pay compensation for damage caused by its space object on the surface of the earth or to aircraft in flight. The important content of the art. 2 is the responsible entity is the 'State' not the 'Company'. According by Korean Space Exploration Act art. 14, person who launches space objects according to art. 8 and art. 11 must bear the liability for damages owing to space accidents of the space objects. Could Korean government apply the Products Liability Act which is enter into force from July 1, 2002 to space launching person? And what is the contact type between Korea Aerospace Research Institute(KARl) and Russia manufacturer. Is that a Co-Development contract or Licence Product contract? And there is no exemption clause to waive the Russia manufacturer's liability which we could find it from other similar contract condition. If there is no exemption clause to the Russia manufacturer, could we apply the Korean Products Liability Act to Russia one? The most important legal point is whether we could apply the Korean Products Liability Act to the main component company. According by the art. 17 of the contract between KARl and the company, KARl already apply the Products Liability Act to the main component company. For reference, we need to examine the Appalachian Insurance co. v. McDonnell Douglas case, this case is that long distance electricity communication satellite of Western Union Telegraph company possessions fails on track entry. In Western Union's insurance company supplied to Western Union with insurance of $ 105 millions, which has the satellite regard as entirely damage. Five insurance companies -Appalachian insurance company, Commonwealth insurance company, Industrial Indemnity, Mutual Marine Office, Northbrook Excess & Surplus insurance company- went to court against McDonnell Douglases, Morton Thiokol and Hitco company to inquire for fault and strict liability of product. By the Appalachian Insurance co. v. McDonnell Douglas case, KARl should waiver the main component's product liability burden. And we could study the possibility of the adapt 'Government Contractor Defense' theory to the main component company.

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Characterization of Lactate Dehydrogenase in Acanthogobius hasta (풀망둑(Acanthogobius hasta) 젖산탈수소효소의 특성)

  • Yum, Jung-Joo
    • Journal of Life Science
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    • v.18 no.2
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    • pp.264-272
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    • 2008
  • The lactate dehydrogenase (EC 1.1.1.27, LDH) isozymes in tissues from Acanthogobius hasta were characterized by biochemical, immunochemical and kinetic methods. The activities of LDH in skeletal muscle and eye tissues were 65.30 and 53.25 units, but LDH activities in heart and liver tissues were very low. LDH/CS (EC 4.1.3.7, citrate synthase) in skeletal muscle was the highest as 22.29. Specific activities of LDH in brain, eye and skeletal muscle were 56.45, 38.04 and 11.0 units/mg, respectively. The LDH isozymes in tissues were separated by polyacrylamide gel electrophoresis after immunoprecipitation with antiserum against $A_4,\;B_4$ eye-specific $C_4$ and liver-specific $C_4$. LDH $AC_4$ isozymes were detected predominantly in skeletal muscle, brain and eye tissues, and $B_4$ isozyme was detected in heart. Anodal eye-specific $C_4$ and cathodal liver-specific $C_4$ were coexpressed in A. hasta. The eye-specific $C_4$ isozyme showed higher activity in eye tissue, but liver-specific $C_4$ isozyme showed lower activity in liver. As a result, one part of molecular structures in $A_4\;and\;C_4,\;A_4\;and\;B_4$, and eye-specific $C_4$ and liver-specific $C_4$ were similar, but in $B_4\;and\;C_4$ were different with each other. Therefore the subunit A may be conservative in evolution, and the evolution of subunit B seems to be faster than that of subunit A. The LDH $A_4$ isozyme of skeletal muscle was purified in the fraction from elution with NAD+ containing buffer of affinity chromatography and eye-specific $C_4$ isozyme was eluted right after $A_4$, so the structure of eye-specific $C_4$ isozyme is similar to $A_4$. And LDH activity remained 35.22-43.47% as a result of the inhibition by pyruvate, the Michaelis-Menten constant values for pyruvate was 0.080-0.098 mM, and Vmax were 153.85 units, 35.09 units in skeletal muscle and eye, respectively. Also the $B_4$ isozyme was the thermo-stablest and $C_4$ was stabler than $A_4$ isozyme. The optimum pH of LDH was 6.5. The results mentioned above indicate that isozymes in tissues showed the properties between LDH $A_4\;and\;B_4$ isozyme as A. hasta was adapted to hypoxic conditions. Also LDH seems to function more effectively under anaerobic condition because LDH in skeletal muscle and eye tissues have high affinity for pyruvate.

Effect of Small Black Soybean Powder on Blood Glucose and Insulin Sensitivity in Streptozotocin-Induced Diabetic Rats (Streptozotocin-유도 당뇨모델을 이용한 쥐눈이콩 분말의 혈당강하 및 인슐린 감수성의 효과)

  • Lee, Dae-Hoon;Kwak, Dong-Hoon;Kim, Sung-Min;Ju, Eun-Jin;Choi, Han-Gil;Kim, Ok-Hee;Hwang, Jin-Bong;Bae, Nahm-Gung;Jung, Kyu-Yong;Han, Jin-Chul;Park, Hum-Dai;Choo, Young-Kug
    • Journal of the Korean Society of Food Science and Nutrition
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    • v.33 no.10
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    • pp.1618-1625
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    • 2004
  • Beans are acknowledged to be food resources, which have more abundant proteins and fats. The constituent parts of beans (i.e. aspartic aid, glycine, arginine) are effective against diabetes, and dietary fiber contained in the beans has an important property to maintain insulin sensitivity. Based on these, using streptozotocin (STZ)-induced diabetic rats, this study examined how the rat-eye soybean, which is principal products of the Imsil province, is effective to attenuate and/or prevent the development of diabetes mellitus. We divided rats into the non-diabetic and diabetic group, and diabetic group was further subdivided into six experimental groups [DC, diabetic control; DI, diabetes with insulin treatment (4∼6 IU/rat); DB, diabetes with black bean; DY, diabetes with yellow soybean; DS, diabetes with rat-eye soybean; DSS, diabetes with vinegar-fermented rat-eye soybean. All bean treatment (1.5 mg/l g body weight).]. Food efficiency ratio (FER), body weight and insulin sensitivity in diabetic rats were significantly reduced compared to those in normal control animals. These reductions were obviously attenuated by administration of a variety of beans used in this study (20∼30%), and the recovery effects were comparable to the results obtained by insulin treatment. Taken together, this study suggests that all beans used may have an essential property to improve and/or attenuate the development of diabetes mellitus in rats.

A Study on the Actual Conditions of Smoking in Middle and High School Students in One Region (일 지역 중·고등학생의 흡연실태)

  • Kim, Hyeon-Ok
    • Journal of the Korean Society of School Health
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    • v.12 no.1
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    • pp.149-167
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    • 1999
  • To investigate the actual conditions of smoking in middle and high school students in Chinan County, I used a sturctured questionnaire for 1,579 students attending twelve middle-high schools from December 1, 1998 to December 20, 1998. I collected and data correlated the using an $SPSS-PC^+$ 1. The smoking rate of middle-high schoo1 students in Chinan County was 17.9%, relatively high. This smoking rate was different according to the gender, grade, religion, and economic situation. In mals, high school students, non-religious, students low income family students, the smoking rate was higher. The smoking rate of high school students was almost the same as the smoking rate of adults, generally higher than that of foreign teenagers. Because the smoking rat of studinets in the third grade of middle school and in the first grade of high School was six times higher, increased education should be conducted during this time in an attempt to curb the sudden increase of the smoking rate. The smoking rate of girl students was 5.0%; this has increased mor than three times from ten years ago. Consequently, counter measures should be taken against the smoking of female students as well as juvenile smoking in general. In addition, the smoking rate of middle-high school students showed interesting differences when correnated with enviornmental factors. Students with low grades, who are not satisfied with school life, who don't have both parents, who have uncaring parents who nare too strict or too arbitary, who have smoking parents, or who have experienced smoking commonly smoked. Therefore, to lower the smoking rate we should improve the school environment, improve a student's interest in school life. And parents or siblings should lead by example and quit smoking at home. Schools should educate students more effectively concerning the harmful effects of smoking and create an accurate understanding of its dancers. From the beginning, we should teach students never ever to touch cigaretts. 2. The surve discovered that most students started out of curiosity, or solicitantion from friends or elders at middle school, and had been smoking one to five cigarettes for more than a year. They obtained cigarettes at stores and most of them have friends who smoke. As a result anti-smoking education should be conducted at elementary schools prior to middle school. More than 95% of the teenagers who smoke had friends who smote and smoked out of curiosity or the recommendation of elders. Thus, we must focus on teenagers who smoke in group, rather than individually. Fuyrthermore, the strict application of the regulation of tobacco sales as well as tobacco cooperation from retailers are needed. While students did not show any mood or academic achievement difference after beginning smoking, 58.1% of the students a health situdation that was worse. Juvenile smoking is more harmful to the juvenile than adult smoking is to the adult. This should be focused on in an anti-smoking campaign. 3. Students who smoke hada more positive attitude toward smoking than students who don't smoke. Students who smoke had a tendency to have a nuetral position and are not concerned about smoking compared to non-smoking students. The survey showed that the great number of students had a nuetral position. Because this nuetrality may increase Juvenile smoking, education that provides an exact understanding of smoking should be performed to build the correct attidude toward smoking. 4. Middle school students smoke when angry, gloomy, anxious, a lone and when they have some problems to solve, on when they feel inconveniened in other wores, they smoke to reliver stress. They also smok due to addiction. Because smoking is not a praetical method to relieve stress, a program which helps to acquire positive relief stress should be provided to help reduce smoking. 5. About 65% of students who smoke want to quit smoking because of health problem, 78% of them have tried mor than once to quit but failed due to weak will power and peer pressure from friends who smoke. Juvenile smoking is group, oriented. Thus, the program that advances less smoking will be the one that focuseds on groups. 6. As for advice to students who want to quit smoking, "persuasion" was used most commonly, followed by a "presentation on how to quit smoking". Another method were severe punishment. About 70% of the students wanted the anti-smoking guide at school. 7. Most students (73.5%) had a position that more anti-smoking education at school is needed. Obriously, then, anti-smoking education at middle-high schools should be reinfoced. Although the education which explains the harmful influence of tobacco is known as an efficient way prevent smoking; it does not influence students who already smoke. Therefore, for students who smoke, multi-dimensional approaches must be attempted that include physical training, phychokogical approache, consultation and discussion, medical chek-ups, audio-visual education technigues, and professonal instructors, in addition, because smoking students have more negative on lukewarm attitude to anti-smoking education anti-smoking education should be conducted through a communicative style by dedicated teachers who care about students. In order to increase the effectiveness of this program.

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Immunocytochemistry of Metallothionein Expression in Developing Rat Liver (발생중인 흰쥐 간에서의 Metallothionein 발현에 관한 면역세포화학적 연구)

  • Oh, Seung-Han;Ahn, Young-Mo;Shin, Kil-Sang;Kim, Wan-Jong
    • Applied Microscopy
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    • v.34 no.3
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    • pp.171-178
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    • 2004
  • Metallothionein (MT) is a family of ubiquitous, low molecular weight (6-7 kDa), cysteine-rich protein with a high affinity to metal ions and has no aromatic amino acids and histidine. Some of the known functions of MT include detoxification of heavy metals and alkylating agents and neutralization of free radicals. Also, this protein may affect a number of cellular processes including gene expression, apoptosis, proliferation and differentiation. But, its actual functions are still not clear. The present study was undertaken to examine immunocytochemically the localization of MT in developing rat liver. On the day 11 of gestation, the fetal rat liver has already been formed and contained numerous oval cells with high nuclear cytoplasmic ratio, which were the progenitors of hepatic parenchymal cells, but no reaction products of MT were detected at this time. And then, positive reactions against MT started to appear predominantly in the parenchymal cells of liver from the 13th day after gestation. Reaction products, immunogold particles or brown coloration, were localized at both the nucleus and the cytoplasm of the parenchymal cells, except mitochondria. The intensity of this reaction gradually increased, and exhibited the strongest at birth. The intensity of MT staining and immunogold labelling diminished with growth, and by the 15th day after birth weak positive reaction was observed in the cells. In brief, positive reactions for MT were observed in the oval cells and the parenchymal cells during fetal stage, meanwhile they were present only in the parenchymal cells after birth. The present results suggest that MT possibly involves parechymal cell proliferation and differentiation through the storage or the supply of various metal ions in the developing rat liver.

Development of Neural Network Based Cycle Length Design Model Minimizing Delay for Traffic Responsive Control (실시간 신호제어를 위한 신경망 적용 지체최소화 주기길이 설계모형 개발)

  • Lee, Jung-Youn;Kim, Jin-Tae;Chang, Myung-Soon
    • Journal of Korean Society of Transportation
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    • v.22 no.3 s.74
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    • pp.145-157
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    • 2004
  • The cycle length design model of the Korean traffic responsive signal control systems is devised to vary a cycle length as a response to changes in traffic demand in real time by utilizing parameters specified by a system operator and such field information as degrees of saturation of through phases. Since no explicit guideline is provided to a system operator, the system tends to include ambiguity in terms of the system optimization. In addition, the cycle lengths produced by the existing model have yet been verified if they are comparable to the ones minimizing delay. This paper presents the studies conducted (1) to find shortcomings embedded in the existing model by comparing the cycle lengths produced by the model against the ones minimizing delay and (2) to propose a new direction to design a cycle length minimizing delay and excluding such operator oriented parameters. It was found from the study that the cycle lengths from the existing model fail to minimize delay and promote intersection operational conditions to be unsatisfied when traffic volume is low, due to the feature of the changed target operational volume-to-capacity ratio embedded in the model. The 64 different neural network based cycle length design models were developed based on simulation data surrogating field data. The CORSIM optimal cycle lengths minimizing delay were found through the COST software developed for the study. COST searches for the CORSIM optimal cycle length minimizing delay with a heuristic searching method, a hybrid genetic algorithm. Among 64 models, the best one producing cycle lengths close enough to the optimal was selected through statistical tests. It was found from the verification test that the best model designs a cycle length as similar pattern to the ones minimizing delay. The cycle lengths from the proposed model are comparable to the ones from TRANSYT-7F.

Hypoglycemic Effect of Culture Broth of Bacillus subtilis S10 Producing 1-Deoxynojirimycin (1-Deoxynojirimycin을 생산하는 Bacillus subtilis S10 배양액의 혈당강하 효과)

  • Cho, Yong-Seok;Park, Young-Shik;Lee, Jae-Yeon;Kang, Kyung-Don;Hwang, Kyo-Yeol;Seong, Su-Il
    • Journal of the Korean Society of Food Science and Nutrition
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    • v.37 no.11
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    • pp.1401-1407
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    • 2008
  • 1-Deoxynojirimycin (DNJ) is a strong $\alpha$-glucosidase inhibitor which inhibits hyperglycemia in animals. To select the Bacillus strains highly producing DNJ, 4,000 strains were isolated from soil and grain samples. By the inhibitory activity against $\alpha$-glucosidase, nine Bacillus strains were selected and then identified by 16S rDNA sequencing. B. subtilis S10 was finally selected as the best strain for the production of DNJ. Various carbon sources and nitrogen sources in culture medium were evaluated for the highest production of DNJ. As the results, the optimized concentration of carbon source and nitrogen source was 1.0% galactose and 1.6% polypeptone and the concentration of DNJ produced was 0.75 g/L. The effect of culture supernatant of B. subtilis S10 on lowering blood glucose level was investigated in streptozotocin (STZ)-induced diabetic mice model. Mice were randomly assigned to control group (saline) and three test groups such as acarbose group, silkworm powder group and B. subtilis S10 group. After eight-week oral feeding, blood glucose levels of the B. subtilis S10 and silkworm powder groups were respectively $209.1{\pm}19.6\;mg/dL$ (59.1%) and $208.6{\pm}39.8\;mg/dL$ (59.0%) lower than $510{\pm}10\;mg/dL$ of the control group. These results indicated that the culture supernatant of B. subtilis S10 was able to reduce the blood glucose level in STZ-induced diabetic mice.

MECHANISM IN ANTIBACTERIAL ACTIVITY OF POLYPHOSPHATES AGAINST PORPHYROMONAS ENDODONTALIS (Porphyromonas endodontalis에 대한 Polyphosphate의 항균기전에 관한 연구)

  • Choi, Sung-Baik;Park, Sang-Jin;Choi, Gi-Woon;Choi, Ho-Young
    • Restorative Dentistry and Endodontics
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    • v.25 no.4
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    • pp.561-574
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    • 2000
  • Poly-P has been used to prevent decomposition of foods and has been shown to have inhibitory effect on the growth of gram positive bacteria. The purpose of this study was to evaluate the effect of poly-P on the growth of Porphyromonas endodontalis, a gram negative obligate anaerobic rod, endodontopathic bacterium. P. endodontalis ATCC 35406 was in BHI broth containing hemin and vitamin K with or without poly-P. Inhibitory effect of each poly-P which was added at the beginning(lag phase) or during(exponential phase) the culture, MIC(minimum inhibitory concentration) was determined by measuring the optical density of the bacterial cell at 540nm. Viable cell counts were measured to determined whether poly-P has a bactericidal effect. Leakage of intracellular nucleotides from P. endodontalis was determined at 260nm and morphological change of P. endodontalis was observed under the TEM(transmission electron microscope). Binding of 32P-labeled poly-P to P. endodontalis was examined. SDS-polyacrylamide gel electrophoresis and zymography were performed to observe the changes in protein and enzyme profiles of P. endodontalis, respectively. The results from this study were as follows : 1. The minimal inhibitory concentration(MIC) of poly-P to P. endodontalis appeared to be 0.04~0.05%. 2. Poly-P added to the P. endodontalis culture during the exponential phase of P. endodontalis was as much effective as poly-P added at the begining of the culture, suggesting that the antibacterial effect of poly-P is not much dependent on the initial inoculum size of P. endodontalis. 3. Poly-P are bactericidal to P. endodontalis, demonstrating the decrease of the viable cell counts. 4. Intracellular nucleotide release from the P. endodontalis, was not increased in the presence of poly-P and was not reversed by the addition of divalent cations like $Ca^{2+}$ and $Mg^{2-}$. 5. Under the TEM, it was observed that fine electro-dense materials were prominent in the poly-P grown P. endodontalis, appearing locally in the cell, and the materials were more abundant and more dispersed in the cell as the incubation time with poly-P increased. In addition, highly electron dense granules accumulated in many poly-P grown cells, most of which were atypical in their shape. 6. Binding of 32P-labeled poly-P to P. endodontalis appeared to be 32.8 and 45.5 and 53.4% at 30 minutes, 1 hours and 2 hours, respectively. 7. In the presence of poly-P. the synthesis of proteins with apparent molecular masses of 25, 27, 35, 45 was lost or drastically decreased whereas expression of a protein with an apparent molecular mass of 75 was elevated. 8. Proteolytic activity of P. endodontalis was decreased by poly-P. The overall results suggest that use of poly-P may affect the growth of P. endodontalis, and the anti-bacterial activity of poly-P seems largely bactericidal. Changes in shape, protein expression, and proteolytic activity of P. endodontalis by poly-P may be directly and indirectly attributed to the antibacterial effect of poly-P. Further studies will be needed to confirm the effect of poly-P.

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Effect of X-Irradiation on the Levels of some Sulfhydryl Groups, Protein and Cell Volume of Ehrlich Ascites Tumour Cells (X-선(線) 조사(照射)가 Ehrlich 암세포(癌細胞)의 용적(容積), 단백양(蛋白量) 및 수종(數種) Sulfhydryl 기(基)에 미치는 영향(影響)에 관(關)하여)

  • Yu, Choon-Shik;Choo, Young-Eun
    • The Korean Journal of Physiology
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    • v.3 no.2
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    • pp.9-16
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    • 1969
  • It is well known that a number of -SH and -SS containing substances afford a certain measure of protection against radiation effects in many biological systems, and it is conceivable that inherent -SH levels in Ehrlich ascites tumour (ELD)cells may be of decisive improtance with respect to the development of cellular radiation injury. So far, little effort has been directed to elucidate the changes in levels of different -SH and -SS groups in ELD cells when the tumour-bearing whole animal was subjected to the sublethal dose of X-irradiation. The present study was designed to bring some lights in the possible changes of and relationship between various sulfhydryl levels, such as P-SH, NP-SH and NP-SS, as well as the content of protein and cell volume of ELD cells, after subjecting the ELD mice to 1,200 r of X-irradiation. The animals used in this experiment were all mixed bred mice of $20{\sim}25\;gm$ in body weight (approximately 2 months old) irrespective of sex. 12 mice in one experiment were inoculated intraperitoneally with 0.2 ml of ascites tumour cells $(2{\times}10^6\;cells)$, and on the 7th day of the tumour growth, they were X-irradiated with 1,200 r, using the conventional X-ray machine under the following conditions: 200 Kv at 15 mA, 0.5 mm Cu filter, target-skin distance: 50 cm. Radiation dose was measured with the the Philip integrating dosimeter. At 24, 36, 48 and 60 hours after the X-irradiation, the mice were killed by cervical dislocation, and the tumours were taken out. Freshly withdrawn ascites tumours were placed in ice, and immediately the cell concentration was measured with the Coulter Cell Counter (Model B), and the hematocrit of the tumour cells were also determined. Cell volume was thus calculated by the cell concentration and hematocrit value. P-SH content of ELD cells was measured potentiometrically according to the method of Calcutt & Doxey, and NP-SH and NP-SS contents were measured spectrophotometrically by the method described by Ellman. Protein content of ELD cells was determined with the Folin phenol reagent by Lowry et al. Altogether, 48 experimental mice were used, and 12 mice with the only exception of X-irradiation were used as the control. Results obtained indicate that the contents of all the cellular sulfhydryl groups as well as cell volume and protein content of the ELD cells increase significantly as time progresses after the sub-lethal X-ray dose of 1,200 r was given and that all the increase is in a lineal fashion. The regression lines of the relative values, (i. e., taking each control value as 1) of all the values obtained, and the regression lines of cell volume, protein and NP-SH are identical, whereas those of NP-SS and P-SH appear to be widely seperated. However, the difference of those two lines (NP-SS & P-SH) were found to be not significant statistically (p>0.05). Therefore, it can be concluded from the above results that all the values examined increase in a lineal fashion with no statistically significant difference among them. Also, with the radiation dose of 1,200 r, the ELD cell becomes enlarged and swollen progressively up to 60 hours post-irradiation and it becomes more than two times of the original normal size at 60 hours after the irradiation, and up to this stage, it seems apparent that the cell division has been slow due to the X-irradiation applied in this experiment. It is well understandable that the contents of NP-SH, NP-SS, P-SH and protein of the ELD cells increase in parallel with the increase of the cell volume by the X-ray does used, but it also seems interesting to note that all the cellular substances tested show no appreciable difference in the pattern of increase.

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