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Characterization of Homocysteine ${\gamma}$-Lyase from Submerged and Solid Cultures of Aspergillus fumigatus ASH (JX006238)

  • El-Sayed, Ashraf S.;Khalaf, Salwa A.;Aziz, Hani A.
    • Journal of Microbiology and Biotechnology
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    • v.23 no.4
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    • pp.499-510
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    • 2013
  • Among 25 isolates, Aspergillus fumigatus ASH (JX006238) was identified as a potent producer of homocysteine ${\gamma}$-lyase. The nutritional requirements to maximize the enzyme yield were optimized under submerged (SF) and solid-state fermentation (SSF) conditions, resulting in a 5.2- and 2.3-fold increase, respectively, after the last purification step. The enzyme exhibited a single homogenous band of 50 kDa on SDS-PAGE, along with an optimum pH of 7.8 and pH stability range of 6.5 to 7.8. It also showed a pI of 5.0, as detected by pH precipitation with no glycosyl residues. The highest enzyme activity was obtained at $37-40^{\circ}C$, with a $T_m$ value of $70.1^{\circ}C$. The enzyme showed clear catalytic and thermal stability below $40^{\circ}C$, with $T_{1/2}$ values of 18.1, 9.9, 5.9, 3.3, and 1.9 h at $30^{\circ}C$, $35^{\circ}C$, $40^{\circ}C$, $50^{\circ}C$, and $60^{\circ}C$, respectively. Additionally, the enzyme $K_r$ values were 0.002, 0.054, 0.097, 0.184, and 0.341 $S^{-1}$ at $30^{\circ}C$, $35^{\circ}C$, $40^{\circ}C$, $50^{\circ}C$, and $60^{\circ}C$, respectively. The enzyme displayed a strong affinity to homocysteine, followed by methionine and cysteine when compared with non-S amino acids, confirming its potency against homocysteinuria-related diseases, and as an anti-cardiovascular agent and a specific biosensor for homocysteinuria. The enzyme showed its maximum affinity for homocysteine ($K_m$ 2.46 mM, $K_{cat}\;1.39{\times}10^{-3}\;s^{-1}$), methionine ($K_m$ 4.1 mM, $K_{cat}\;0.97{\times}10^{-3}\;s^{-1}$), and cysteine ($K_m$ 4.9 m M, $K_{cat}\;0.77{\times}10^{-3}\;s^{-1}$). The enzyme was also strongly inhibited by hydroxylamine and DDT, confirming its pyridoxal 5'-phosphate (PLP) identity, yet not inhibited by EDTA. In vivo, using Swiss Albino mice, the enzyme showed no detectable negative effects on platelet aggregation, the RBC number, aspartate aminotransferase, alanine aminotransferase, or creatinine titer when compared with negative controls.

A Study on Melting Phenomena of Solder Paste (솔더 페이스트의 용융현상 연구)

  • 김문일;안병용;정재필
    • Journal of the Microelectronics and Packaging Society
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    • v.8 no.1
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    • pp.5-11
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    • 2001
  • Melting behavior and bridge phenomenon of solder paste, which is essential for surface mount technology in pachaging, were investigated. Solder paste of Sn-37%Pb was printed on Cu-pattern of PCB, and heated over melting point. Melting behavior of the paste was observed using CCD-camera. In order to modelize the melting and agglomeration phenomena of paste, two solder balls of 0.76 mm diameter were used. As experimental results, the paste start to melt from the margin of the printed shape. The height of the melted paste decreased from 270 $\mu\textrm{m}$ to 200 $\mu\textrm{m}$ firstly, and finally recovered to 250 $\mu\textrm{m}$ During the melting procedure, pores were evolved from the molted paste. Concerning melting model of solder ball, relationship between contact area of solder ball and soldering time was derived as $\chi^2/t=4r \; \gamma/\eta=7.56 m^2$/s at $280^{\circ}C$.

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Characterization and Expression Profile Analysis of a New cDNA Encoding Taxadiene Synthase from Taxus media

  • Kai, Guoyin;Zhao, Lingxia;Zhang, Lei;Li, Zhugang;Guo, Binhui;Zhao, Dongli;Sun, Xiaofen;Miao, Zhiqi;Tang, Kexuan
    • BMB Reports
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    • v.38 no.6
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    • pp.668-675
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    • 2005
  • A full-length cDNA encoding taxadiene synthase (designated as TmTXS), which catalyzes the first committed step in the Taxol biosynthetic pathway, was isolated from young leaves of Taxus media by rapid amplification of cDNA ends (RACE). The full-length cDNA of TmTXS had a 2586 bp open reading frame (ORF) encoding a protein of 862 amino acid residues. The deduced protein had isoelectric point (pI) of 5.32 and a calculated molecular weight of about 98 kDa, similar to previously cloned diterpene cyclases from other Taxus species such as T. brevifolia and T. chinenisis. Sequence comparison analysis showed that TmTXS had high similarity with other members of terpene synthase family of plant origin. Tissue expression pattern analysis revealed that TmTXS expressed strongly in leaves, weak in stems and no expression could be detected in fruits. This is the first report on the mRNA expression profile of genes encoding key enzymes involved in Taxol biosynthetic pathway in different tissues of Taxus plants. Phylogenetic tree analysis showed that TmTXS had closest relationship with taxadiene synthase from T. baccata followed by those from T. chinenisis and T. brevifolia. Expression profiles revealed by RT-PCR under different chemical elicitor treatments such as methyl jasmonate (MJ), silver nitrate (SN) and ammonium ceric sulphate (ACS) were also compared for the first time, and the results revealed that expression of TmTXS was all induced by the tested three treatments and the induction effect by MJ was the strongest, implying that TmTXS was high elicitor responsive.

Effects of Water Temperature, Salinity and Irradiance on the Growth of the Toxic Dinoflagellate, Gymnodinium catenatum (Graham) Isolated from Yeosuhae Bay, Korea (여수해만에서 분리한 유독 와편모조류, Gymnodinium catenatum (Graham)의 성장에 미치는 수온, 염분과 광 조건)

  • Oh, Seok-Jin;Yoon, Yang-Ho
    • ALGAE
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    • v.19 no.4
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    • pp.293-301
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    • 2004
  • A chain-forming toxic din flagellate, Gymnodinium catenatum (Graham) was known as a paralytic toxin-producer among Gymnodinoid group. In the study, the effects of water temperature, salinity and irradiance on the growth of G. catenatum isolated from Yeosuhae Bay, Korea were investigated. Water temperature range in which G. catenatum showed specific growth rate higher than 0.3 day$^{-1}$ were above about 18${^{\circ}C}$. However, salinity did not have such an effect on growth of G. catenatum. The maximum growth rate (0.5 day$^{-1}$) was obtained at 25${^{\circ}C}$ and 30 psu. The specific growth rate (u) expressed as a polynomial equation as functions of temperature (T; ${^{\circ}C}$) and salinity (S; psu) was $\mu$ = 0.005·T$^2$ - 0.0001164 T$^3$ - 0.063-S + 0.005-S$^2$ - 0.00007608-S$^3$ - 0.003-T-S + 0.00005308-T$^2$-S. Thus, in aspects of water temperature and salinity, the species may be expected to survive in most Korean coastal waters from early summer to autumn. The irradiance-growth curve was described as = 0.16 (I - 10.4)/(1 + 21.8) at 18${^{\circ}C}$ and 30 psu, indicating a half-saturation (Ks) photon flux density (PFD) of 42.6$\mu$mol m$^{-2}s^{-1}$ and compensation PFD (I$_0$) of 10.4$\mu$mol m$^{-2}s^{-1}$. These characteristic responses to irradiance suggest that G. catenatum can reside at the sub-surface.

Effects of Kojic acid, Arbutin and Vitamin C on cell viability and melanin synthesis in B16BL6 cells

  • Park, Yumi;Lee, Jongsung;Park, Junho;Park, Deokhoon
    • Journal of the Society of Cosmetic Scientists of Korea
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    • v.29 no.1
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    • pp.151-167
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    • 2003
  • Melanin biosynthesis is a human defense mechanism to protect skin from UV irradiation and also determines colors of hair and skin. However, as a interest on skin-whitening increases, researches to prevent pigmentation and hypersynthesis of melanin in skin are being actively in progress. Active components used as a whitening agent in cosmeceuticals are kojic acid, arbutin, vitamin C and hydroquinone. However, until now, because comparison researches among them in the aspect of both melanin formation and cellular toxicity have not been performed, we can't exactly estimate merits and defects of them as a whitening agent. To this end, we performed experiments to compare their effects on cell viability and melanin formation. As a first step, in vitro tyrosinase inhibition assay was done. While kojic acid and hydroquinone showed strong inhibition activities(their IC$\_$50/s are all < 100uM), arbutin and vitamin C showed weak activities. IC$\_$50/s of arbutin and vitamin C are 100uM and 400∼500uM, respectively. In B16BL6 melanoma cells, like in vitro tyrosinase inhibition assay, arbutin and kojic acid showed more strong inhibition effect on melanin synthesis than vitamin C. And unlike arbutin, vitamin C and kojic acid induced cell death at high concentration. Although arbutin showed no cytotoxicity, it has side effect to induce morphological change at high concentration.. In this paper, we suggest both kojic acid and arbutin have stronger ability to inhibit melanogenesis than vitamin C. And they also have side effect, that is, kojic acid induces cell death like vitamin C and arbutin changes cell morphology respectively.

Activities of E6 Protein of Human Papillomavirus 16 Asian Variant on miR-21 Up-regulation and Expression of Human Immune Response Genes

  • Chopjitt, Peechanika;Pientong, Chamsai;Bumrungthai, Sureewan;Kongyingyoes, Bunkerd;Ekalaksananan, Tipaya
    • Asian Pacific Journal of Cancer Prevention
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    • v.16 no.9
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    • pp.3961-3968
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    • 2015
  • Background: Variants of human papillomavirus (HPV) show more oncogenicity than do prototypes. The HPV16 Asian variant (HPV16As) plays a major role in cervical cancer of Asian populations. Some amino acid changes in the E6 protein of HPV16 variants affect E6 functions such as p53 interaction and host immune surveillance. This study aimed to investigate activities of HPV16As E6 protein on modulation of expression of miRNA-21 as well as interferon regulatory factors (IRFs) 1, 3, 7 and c-fos. Materials and Methods: Vectors expressing E6 protein of HPV16As (E6D25E) or HPV16 prototype (E6Pro) were constructed and transfected into C33A cells. HCK1T cells expressing E6D25E or E6Pro were established by transducing retrovirus-containing E6D25E or 16E6Pro. The E6AP-binding activity of E6 and proliferation of the transfected C33A cells were determined. MiR-21 and mRNA of interesting genes were detected in the transfected C33A cells and/or the HCK1T cells, with or without treatment by culture medium from HeLa cells (HeLa-CM). Results: E6D25E showed binding activity with E6AP similar to that of E6Pro. Interestingly, E6D25E showed a higher activity of miR-21 induction than did E6Pro in C33A cells expressing E6 protein. This result was similar to the HCK1T cells expressing E6 protein, with HeLa-CM treatment. The miR-21 up-regulation significantly corresponded to its target expression. Different levels of expression of IRFs were also observed in the HCK1T cells expressing E6 protein. Interestingly, when treated with HeLa-CM, IRFs 1, 3 and 7 as well as c-fos were significantly suppressed in the HCK1T cells expressing E6D25E, whereas those in the HCK1T cells expressing E6Pro were induced. A similar situation was seen for IFN-${\alpha}$ and IFN-${\beta}$. Conclusions: E6D25E of the HPV16As variant differed from the E6 prototype in its activities on epigenetic modulation and immune surveillance and this might be a key factor for the important role of this variant in cervical cancer progression.

Highly (111)-oriented SiC Films on Glassy Carbon Prepared by Laser Chemical Vapor Deposition

  • Li, Ying;Katsui, Hirokazu;Goto, Takashi
    • Journal of the Korean Ceramic Society
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    • v.53 no.6
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    • pp.647-651
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    • 2016
  • SiC films were prepared on glassy carbon substrates by laser chemical vapor deposition under a high pressure of $10^4Pa$ using a diode laser (wavelength = 808 nm) and a polysilaethylene precursor. (111)-oriented SiC films were formed at a deposition temperature ($T_{dep}$) range of 1150 - 1422 K. At $T_{dep}=1262K$, the SiC film with a high Lotgering factor of above 0.96 showed an exhibited pyramid-like surface morphology and flower-like grains. The highest deposition rate ($R_{dep}$) was $220{\mu}m\;h^{-1}$ at $T_{dep}=1262K$.

Medicinal Herbal Complex Extract with Potential for Hair Growth-Promoting Activity (발모효과를 가지는 한방복합처방단)

  • Lee, Jun Young;Im, Kyung Ran;Jung, Taek Kyu;Lee, Myoung-Hee;Yoon, Kyung-Sup
    • Journal of the Society of Cosmetic Scientists of Korea
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    • v.38 no.4
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    • pp.277-287
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    • 2012
  • To develop new therapeutic materials to prevent hair loss and enhance hair growth, we developed a medicinal herbal complex extract (MHCE) using 23 herbs traditionally used in oriental medicine. Medicinal Herbal complex extract was consist of Angelica gigas Nakai, Psoralea corylifolia Linne, Biota orientalis Endlicher, and Eclipta prostrata Linne, Rehmannia glutinosa Liboschitz var. purpurea Makino, Ligustrum lucidum Aiton, Polygonum multiflorum Thunberg, and Sesamum indicum Linne, Sophora angustifolia Sieboldet Zuccarini, Angelica dahurica Benthamet Hooker, and Leonurus sibiricus Linne, Salvia miltiorrhiza Bunge, Prunus persica Batsch, Commiphora molmol Engler, Chrysanthemum indicum Linne, Boswellia carterii Birdwood, Panax ginseng C. A. Meyer, Cnidium officinale Makino, Albizia julibrissin Durazzini, and Corydalis ternata Nakai that have traditionally been used for treating hair loss, preventing gray hair, anti-inflammation, and blood circulation in oriental medicine. In addition, we examined the hair growth effect of MHCE in vitro and in vivo. In vitro, we evaluated the effects of MHCE on cultured HFDPC, HaCaT cells, and murine embryonal fibroblasts (NIH3T3 cells). Also, we evaluated the ability of MHCE to prevent gray hair on murine melanoma cells (B16F1 cells). The hair growth-promoting effect of MHCE in vitro was also observed in vivo using C57BL/6 mice. Our results showed that MHCE significantly increased the proliferation of HFDPC (175 % proliferation at $50{\mu}g/mL$), HaCaT cells (133 % proliferation at $20{\mu}g/mL$), and NIH3T3 cells (120 % proliferation at $50{\mu}g/mL$). MHCE also showed consistent melanogenesis in B16F1 cells (154 % melanin synthesis at $50{\mu}g/mL$). Moreover, MHCE showed potential for hair growth stimulation in C57BL/6 mice experiments (98 % hair growth area on 4 weeks). These results indicate that MHCE may be a good candidate for promotion of hair growth.

Effect of sequence variations within DNA melting region on the rate of formation of open complexes at $\lambdaP_{R}$ promoter ($\lambdaP_{R}$ 프로모터 열린복합체 형성에 미치는 DNA melting 부위 염기서열의 영향)

  • 정현채;노정혜
    • Korean Journal of Microbiology
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    • v.28 no.1
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    • pp.19-26
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    • 1990
  • To examine the effects of sequence variations near the transcriptional start site on the rate of formation of the open complexes at bacteriophage $\lambda P_{R}$ promoter, two mutant promoters were created by site-specific mutagenesis using synthetic oligonucleotides. Mutant I coatains changes at positions -3 and -4 from TT to CC, thus having a 6-bp long G/C stretch between -10 region and transciptional start site (+1). Mutant II has changes at positions -5 and -6 from GG to AA, thereby having a 9-bp long A/T stretch between positions -11 and -3. Selective filter binding assays were performed to measure the rate of formation of the open complexes between the wild-type or two mutant $P_{R}$ promoters on 664 bp fragments and E. coli RNA polymerase at two temperatures. At 37.deg.C, the wild-type and two mutants showed similar rates for the formation of open complex. The second order rate constant $k_{a}$ and $\tau _{int}$, as determined from the .tau.-plot analysis, were $(6.0\pm0.4)\times10^{6}M^{-1}sec^{-1}$ and $11\pm5$sec, respectively. At 18.deg.C, however, the wild-type and two mutant promoters showed differences in the kinetic parameters. k for the wild-type promoter was (2.2$\pm$0.1)\times 10^{6}M^{-1}sec^{-1}$ and $\tau _{int}$ was 76$\pm$sec. Mutant I and II exhibited differences mainly in the rate of isomerization ($\tau_{int,I}=91\pm$10 sec, int,II=34$\pm$ sec), whereas the second order rate constant $k_{a}$ was similar to the wild type value. This result implies that at $18^{\circ}C$, the isomerization rate is determined by both protein conformational change and DNA melting, which are separable kinetically according to the 3-step mechanism of Roe et al.(1984,1985), and that the base changes affected mainly the rate of DNA melting as predicted.lting as predicted.

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