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Cloning and Expression of Isocitrate Lyase, a Key Enzyme of the Glyoxylate Cycle, of Candida albicans for Development of Antifungal Drugs

  • SHIN DONG-SUN;KIM SANGHEE;YANG HYEONG-CHEOL;OH KI-BONG
    • Journal of Microbiology and Biotechnology
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    • v.15 no.3
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    • pp.652-655
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    • 2005
  • This paper describes the development of an enzymatic assay system for the identification of inhibitors of isocitrate lyase (ICL), one of the key enzymes of the glyoxylate cycle that is considered as a new target for antifungal drugs. A 1.6 kb DNA fragment encoding the isocitrate lyase from Candida albicans ATCC10231 was amplified by PCR, cloned into a vector providing His-Patch-thioredoxin-tag at the N-terminus, expressed in Escherichia coli, and purified by metal chelate affinity chromatography. The molecular mass of the purified ICL was approximately 62 kDa, as determined by SDS-PAGE, and the enzyme activity was directly proportional to incubation time and enzyme concentration. The effects of itaconate-related compounds on ICL activity were also investigated. Among them, itaconic acid, 3-nitropropionate, and oxalate had strong inhibitory activities with $IC_{50}$ values of 5.8, 5.4 and $8.6\;{mu}g/ml$, respectively. These inhibitors also exhibited antifungal activity on YPD agar media containing acetate as a sole carbon source, albeit at high concentration. The results indicate that the C. albicans ICL may be a regulatory enzyme playing a crucial role in fungal growth and is a prime target for antifungal agents.

Gram-Positive Bacterial Species and Antimicrobial Susceptibility Patterns Isolated from Chungbuk Area (최근 충북지역에서 분리된 Gram 양성 세균종과 항생물질의 감수성 양상)

  • 황석연;최원창
    • Biomedical Science Letters
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    • v.5 no.2
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    • pp.213-217
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    • 1999
  • In order to control resistant strains and to properly select the antimicrobial agents, it is of quite importance to know current trends of bacterial species and changing patterns of antimicrobial resistance rates. The authors studied the results of 542 Gram-positive strains among 1,689 strains isolated at Chung-buk National University Hospital in 1996. The frequently isolated Gram-positive microorganisms were Staphylococcus aureus, Streptococcus pneumoniae, Staphylococcus epidermidis and Enterococcus faecalis in descending order. S. aureus showed high resistance to penicillin, gentamicin, and susceptibility to teicoplanin and vancomycin. Coagulase negative Staphylococcus was highly resistant to all of the antibiotics used in this experiment except teicoplanin and vancomycin. Enterococcus were highly resistant to vancomycin, penicillin and tetracycline. MIC of Gram-positive oaganisms was appeared to be zig-zag pattern.

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Development of an experimental model for radiation-induced inhibition of cranial bone regeneration

  • Jung, Hong-Moon;Lee, Jeong-Eun;Lee, Seoung-Jun;Lee, Jung-Tae;Kwon, Tae-Yub;Kwon, Tae-Geon
    • Maxillofacial Plastic and Reconstructive Surgery
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    • v.40
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    • pp.34.1-34.8
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    • 2018
  • Background: Radiation therapy is widely employed in the treatment of head and neck cancer. Adverse effects of therapeutic irradiation include delayed bone healing after dental extraction or impaired bone regeneration at the irradiated bony defect. Development of a reliable experimental model may be beneficial to study tissue regeneration in the irradiated field. The current study aimed to develop a relevant animal model of post-radiation cranial bone defect. Methods: A lead shielding block was designed for selective external irradiation of the mouse calvaria. Critical-size calvarial defect was created 2 weeks after the irradiation. The defect was filled with a collagen scaffold, with or without incorporation of bone morphogenetic protein 2 (BMP-2) (1 ㎍/ml). The non-irradiated mice treated with or without BMP-2-included scaffold served as control. Four weeks after the surgery, the specimens were harvested and the degree of bone formation was evaluated by histological and radiographical examinations. Results: BMP-2-treated scaffold yielded significant bone regeneration in the mice calvarial defects. However, a single fraction of external irradiation was observed to eliminate the bone regeneration capacity of the BMP-2-incorporated scaffold without influencing the survival of the animals. Conclusion: The current study established an efficient model for post-radiation cranial bone regeneration and can be applied for evaluating the robust bone formation system using various chemokines or agents in unfavorable, demanding radiation-related bone defect models.

Study on Antioxidant and Anti-inflammatory Activities of Persicaria tinctoria (쪽의 항산화 및 항염증 활성에 대한 연구)

  • Kim, Soo-Jeung;Jang, Tae Won;Kim, Do-Wan;Park, Jae Ho
    • The Korea Journal of Herbology
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    • v.30 no.6
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    • pp.17-24
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    • 2015
  • Objectives : Persicaria tinctoria belongs to the Polygonaceae family and it has been used as the natural dye traditionally. Also, it is well known that the Persicaria tinctoria is used for treating the following symptoms such as fever, inflammation and edema. The purpose of this study is to investigate the effective source of antioxidants and anti-inflammatory agent from various parts of Persicaria tinctoria.Methods : We investigated the antioxidative and anti-inflammatory properties of the Persicaria tinctoria extracts. Antioxidant activities were measured by 1,1-diphenyl-2- picrylhydrazyl (DPPH), 2, 2'-Azino-bis (3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt (ABTS) radical scavenging activity, Fe2+ chelating activity and Reducing power of Persicaria tinctoria extracts. And its inhibitory effect against oxidative DNA damage was evaluated in non-cellular system using φX-174 RF I plasmin DNA. The anti-inflammatory effect of Persicaria tinctoria was measured by using the inhibitory efficacy for the amount of nitric-oxide (NO) produced in LPS induced RAW264.7 cells.Results : The extracts from stem part showed better DPPH scavenging activity compared to those of the leaf and root extracts. Their IC50s were measured as 7.17, 144.40 and 165.07 ug/ml, respectively. These results were similar to that of ABTS radical scavenging assay and reducing power. Also, Persicaria tinctoria showed the protective effects of DNA damage against oxidative stress and anti-inflammatory effect by suppression of NO production in LPS induced RAW264.7 cells.Conclusions : These results showed that various parts of Persicaria tinctoria can be used as an effective source of antioxidants and anti-inflammatory agents via antioxidative activities and anti-inflammatory effect.

Cortex Mori Extract Induces Cancer Cell Apoptosis Through Inhibition of Microtubule Assembly

  • Hwang, Pyoung-Han;Nam, Sang-Yun;Yi, Ho-Keun;Lee, Jung-Chang;Kim, Jae-Cheol;Song, Chang-Ho;Park, Jin-Woo;Lee, Dae-Yeol;Kim, Jung-Soo
    • Archives of Pharmacal Research
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    • v.25 no.2
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    • pp.191-196
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    • 2002
  • The water extract from the root bark of Cortex Mori (CM, Morus alba L.: Sangbaikpi), a mulberry tree, has been known in Chinese traditional medicine to have antiphlogistic, diuretic, and expectorant properties. In this study, the cytotoxicity of CM against tumor cells and its mechanism was examined . CM exhibited cytotoxic activity on K-562, B38O human leukemia cells and B16 mouse melanoma cells at concentrations of > 1 mg/ml. A DNA fragmentation, PARP cleavage, and nuclear condensation assay showed that those cells exposed to CM underwent apoptosis. The water extract of Scutellarie Radix (SR) was used as a negative control and showed no cytotoxicity in those cells. The flow cytometric profiles of the CM-treated cells were also indicative of apoptosis. However, they did not appear to exert the G1 arrest, which is observed in other tubulin inhibitor agents such as vincristine, taxol. The protein-binding test using Biacore and a microtubule assembly-disassembly assay provided evidence showing that CM bound to the tubulins resulting in 3 markets inhibition of the assembly, but not the disassembly of microtubules. The possible nonspecific effect of the CM extract could be excluded due to the results using SR, which did not affect the assembly process. Overall, the water extract of CM induces apoptosis of tumor cells by inhibiting microtubule assembly.

Studies of plant Tumor induction (III) - Antimicrobial action of some bacteriacidal agents to obtain Bacteria-Free Tumor tissue (식물의 암종유발에 관한 연구 (제 3보) - Bacteria-free 암종조직의 획득에 있어서 항생제의 효과)

  • 이민재;홍순우;최영길
    • Korean Journal of Microbiology
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    • v.5 no.2
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    • pp.97-101
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    • 1967
  • Up to the present time, there are only three methods by which we can obtain bacteria free crown gall tissue. According to some papers related to this field, the first method is based on the works of Braun(53') who maintained infected plants at 46-47'c for several days. But the method has a problem that very few plants can tolerate this temperature. The second method is based on the well known observation that old tumors appear to be bacteria free at least 1 or 2% of the explants. Also this method is known to us as laborious and time consuming. The third method is the one we were using that was attempting to kill the bacteria with bacteriacidal agent such as Antibiotics. In fact., it is reported that almost complete control of crown gall of tomato was obtained by Blanchard('51) when plants were grown in a nutrient containing Aureomycin(20${\mu}g$/ml) following needle puncture with the gall bacteria. We have been engaged in making the experiment by applying solution of Penicillin, Streptomycin and of Chloramphenicol(Succinate free) to find the strong bacteriacidal agent through the method of disc plate, and to confirm the effect of antimicrobial action through the method of plant tissue culture system without possible injury to the host plant. The result of this report is the fact the strongest bacteriacidal agent among the above three Antibiotics was Chloramphenicol(Succinate free 1000 p.p.m). and that there happened no injury to the tissue cultures in a White's 10X media containing 1000 p.p.m. of Chloramphenicol.

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Effect of ${\beta}-sitosterol$ from Pueraria thunbergiana on the Antioxidant Enzyme Activities in HEI-OC1 Cells (갈근으로부터 추출한 ${\beta}-sitosterol$이 HEI-OC1 세포의 항산화 효소 활성에 미치는 영향)

  • Hwan, Ji-Young;Chang, Hye-Soon;Yu, Hyeon-Hee;Moon, Hae-Dalma;Jeon, Byung-Hun;You, Yong-Ouk
    • Journal of Physiology & Pathology in Korean Medicine
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    • v.21 no.4
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    • pp.884-890
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    • 2007
  • A mechanism of hair cell damage caused by noise and ototoxic agents is mediated through generation of free radicals and reactive oxygen species(ROS). It is known that most of animals have defense systems of ROS that protect against ROS, and the cochlea of animals also has ROS defense system, which appear efficient in detoxifying ROS generated under normal condition. This system includes several antioxidant enzymes such as superoxide dismutase(SOD), catalase(CAT), glutathione peroxidase (GPX), and glutathione reductase(GR). The radix of Pueraria thunbergiana(P. thunbergiana) is traditionally prescribed to attenuate the clinical manifestation of inner ear dysfunction and various clinical situations including fevers, gastrointestinal disorders, skin problems, migraine headaches, lowering cholesterol, and treating chronic alcoholism in Oriental Medicine. In the present study, to investigate the protection mechanism of ${\beta}-sitosterol$ from P. thunbergiana on cisplatin cytotoxicity toward HEI-OC1, we measured the effects of ${\beta}-sitosterol$ on activities of SOD, CAT, GPX, and GR in cisplatin treated cells. SOD, CAT, GPX, and GR activities were significantly increased in the presence of 0.001-0.1 ${\mu}g/ml$ of ${\beta}-sitosterol$ compared to the control group. These results indicate that ${\beta}-sitosterol$ protects cisplatin-induced HEI-OC1 cell damage through increasing the antioxidant enzyme system such as SOD, CAT, GPX, and GR.

Effect of Types and Mixing Ratios of Water-Soluble Polymers on In Vitro Release Profile of Sold Dispersion for Acyclovir (아시클로비어 고체분산체의 용해도에 대한 수용성 고분자의 종류 및 배합 비율에 따른 효과)

  • Ahn, Yong-San;Lee, Ha-Young;Hong, Keum-Duck;Jung, Sung-Beum;Cho, Sun-Hang;Rhee, John M.;Lee, Hai-Bang;Khang, Gil-Son
    • Journal of Pharmaceutical Investigation
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    • v.34 no.4
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    • pp.289-297
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    • 2004
  • Acyclovir (ACV) is one of the most effective and selective agents against viruses of the herpes group. Because of low solubility, bioavailability of ACV has shown below 30% with oral dosage form. In our previous study, we reported that the fabrication of solid dispersion of ACV was possible and the solid dispersion of ACV and PVP was the most useful in all samples. In this study, we examined the effect of mixture ratio of polymers (PEG and PVP) to ACV. Solubility of ACV was dramatically increased up to 25 mg/ml in $80^{\circ}C$ distilled water. So water was used as a solvent to eliminate problem of residual solvent. Spray drying method was used for the solid dispersion of ACV as solvent extraction. Different scanning calorimeter was used to check degradation of drug. Polymer carriers were PEG 6,000 and PVP. In summary, ACV-PVP (1:3) showed the best solubility in distilled water.

Effect of Insulin, Transferrin and Platelet-Derived Growth Factor Supplemented to Synthetic Oviduct Fluid Medium on In Vitro Development of Bovine Embryos Matured and Fertilized In Vitro (합성난관배양액에 첨가된 Insulin, Transferrin 및 Platelet-Derived Growth Factor (PDGF)가 소 수정란의 체외발육에 미치는 영향)

  • 이은송
    • Journal of Embryo Transfer
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    • v.12 no.3
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    • pp.283-291
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    • 1997
  • In vitro development of bovine embryos is affected by many factors such as energy substrates, amino acids, and some growth factors. It has been reported that mRNA of insulin, PDGF and their receptors are detected in cow embryos, and that some chelating agents such as EDTA and transferrin have beneficial role on mouse and bovine embryos. The author hypothesized that insulin, transferrin arid PDGF added to a culture medium increase in vitro development of bovine embryos by chelating toxic substance(s) or increasing cell growth and metabolism. Immature oocytes from slaughtered ovaries of Holstein cows and heifers were matured for 24 hours in a TCM199 containing 10% fetal calf serum, FSH, LH and estradiol with granulosa cells in vitro. Matured oocytes were coincubated with sperm for 30 hours in a modified Tyrode's medium (IVF). Embryos cleaved to 2- to 4-cell at 30 hours after IVF were selected and cultured in a 30-$\mu$l drop of a synthetic oviduct fluid medium (SOFM) containing 0.8% BSA, Minimum Essential Medium essential and non-essential amino acids, and insulin, transferrin or PDGF for 9 days. Supplementation of a SOFM with insulin, and /or transferrin did not increase develop-mental rate to expanding and hatching blastocyst of 2- to 4-cell bovine embryos compared with control. The highest developmental rate to hatching blastocyst was shown when PDGF was added at the concentration of 10 ng /ml among the supplementing doses tested in the present study (p<0.05). Addition of PDGF without insulin to a SOFM could not increase embrye development, but combined addition of PDGF with insulin significantly increased (p<0.05) embryo development to hatching blastocyst (50%) compared with control (38%). In conclusion, insulin and PDGF supplemented to a SOFM may act synergistically and have beneficial effect on in vitro development of 2- to 4-cell bovine embryos matured and fertilized in vitro.

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Anti-mutagenic and Anti-septic Effects of $\beta$-glucan from Aureobasidium pullulans SM-2001 (흑효모유래 $\beta$-glucan의 패혈증 치료효과 및 항돌연변이 활성 평가)

  • Ku, Sae-Kwang
    • Korean Journal of Oriental Medicine
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    • v.15 no.3
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    • pp.75-82
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    • 2009
  • Anti-mutagenic and anti-septic effects of $\beta$-1,3/1,6-glucan from Aureobasidium pullulans SM-2001 were evaluated on the on the cyclophosphamide (CPA)-cecal ligation puncture (CLP) and CPA-treated mice. To induce immunosuppression and mutagenicity, 150 and 110 mg/kg of CPA were single intraperitoneally injected at 3 or 1 day before CLP or initial $\beta$-glucan administration. In CLP animals, the cecum was mobilized and ligated below the ileocecal valve, punctured through both surfaces twice with a 22-gauge needle. 125 mg/kg of $\beta$-glucan were dissolved in saline and subcutaneously or orally administered in a volume of 10 ml/kg (of body weight), 4 times, 12 hrs intervals from 6 hrs after CLP or 1 day after second dose of CPA. After treatment of $\beta$-glucan, the mortalities were observed in CPA-CLP model, and the appearance of a micronucleus is used as an index for genotoxic potential in CPA model. As results of CPA-CLP sepsis, all animals (9/9, 100%) in CPA-CLP control were dead within 2 days after CLP. In addition, increase of the number of bone marrow MNPCEs indicated mutagenicity were also observed by treatment of CPA. However, $\beta$-glucan treatment effectively inhibited the mortalities in CPA-CLP, and it also reduced the CPA treatment-related mutagenicity, respectively. These results indicated that $\beta$-glucan has effective anti-septic and anti-mutagenic effects and can be used as an agents for treating sepsis and mutagenicity related to high-dose chemotherapy or radiotherapy. However, further studies should be conducted to observe more detail action mechanisms of it's anti-septic and anti-mutagenic effects.

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