• Title/Summary/Keyword: functional characterization

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Physiological Functions of the COPI Complex in Higher Plants

  • Ahn, Hee-Kyung;Kang, Yong Won;Lim, Hye Min;Hwang, Inhwan;Pai, Hyun-Sook
    • Molecules and Cells
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    • v.38 no.10
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    • pp.866-875
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    • 2015
  • COPI vesicles are essential to the retrograde transport of proteins in the early secretory pathway. The COPI coatomer complex consists of seven subunits, termed ${\alpha}-$, ${\beta}-$, ${\beta}^{\prime}-$, ${\gamma}-$, ${\delta}-$, ${\varepsilon}-$, and ${\zeta}$-COP, in yeast and mammals. Plant genomes have homologs of these subunits, but the essentiality of their cellular functions has hampered the functional characterization of the subunit genes in plants. Here we have employed virus-induced gene silencing (VIGS) and dexamethasone (DEX)-inducible RNAi of the COPI subunit genes to study the in vivo functions of the COPI coatomer complex in plants. The ${\beta}^{\prime}-$, ${\gamma}-$, and ${\delta}$-COP subunits localized to the Golgi as GFP-fusion proteins and interacted with each other in the Golgi. Silencing of ${\beta}^{\prime}-$, ${\gamma}-$, and ${\delta}$-COP by VIGS resulted in growth arrest and acute plant death in Nicotiana benthamiana, with the affected leaf cells exhibiting morphological markers of programmed cell death. Depletion of the COPI subunits resulted in disruption of the Golgi structure and accumulation of autolysosome-like structures in earlier stages of gene silencing. In tobacco BY-2 cells, DEX-inducible RNAi of ${\beta}^{\prime}$-COP caused aberrant cell plate formation during cytokinesis. Collectively, these results suggest that COPI vesicles are essential to plant growth and survival by maintaining the Golgi apparatus and modulating cell plate formation.

Synthesis and Characterization of a Near-Infrared Optical Materials for Shielding Infrared Rays

  • Park Su-Yeol;Sin Seung-Rim;Sin Jong-Il;O Se-Hwa;Jeon Geun
    • Proceedings of the Korean Society of Dyers and Finishers Conference
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    • 2005.05a
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    • pp.213-215
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    • 2005
  • The metal complexes can be influenced not only by the central metal atoms and the substituent groups, but also by the native of the chelating atoms. For example, near-infrared absorbing chromophores were synthesized by the reaction of phenylenediamine derivatives with a solution of pottassium hydroxide followed by the addition of nickel(II) chloride. These dyes provide absorbing infrared light over 780-840 nm with an extinction coefficient of $2.5-6.0{\times}10^4$. By introduction of alkyl, alkoxyl, cyano, and other functional group into the parent dye, these dyes greatly improved the solubility in organic solvent. New near-infrared absorbing donor-acceptor chromophores have been investigated by varying the electron donating and accepting strength of the two halves of the molecule. The cyanine chromophores permit the simplest way of obtaining systems that absorb well into the near-infrared region of the spectrum. Cyanine dyes possess high extinction coefficients that initially increase with Increasing chain length. These chromophores could be useful in near-infrared optical materials.

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Biosorption of Lead(II) by Arthrobacter sp. 25: Process Optimization and Mechanism

  • Jin, Yu;Wang, Xin;Zang, Tingting;Hu, Yang;Hu, Xiaojing;Ren, Guangming;Xu, Xiuhong;Qu, Juanjuan
    • Journal of Microbiology and Biotechnology
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    • v.26 no.8
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    • pp.1428-1438
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    • 2016
  • In the present work, Arthrobacter sp. 25, a lead-tolerant bacterium, was assayed to remove lead(II) from aqueous solution. The biosorption process was optimized by response surface methodology (RSM) based on the Box-Behnken design. The relationships between dependent and independent variables were quantitatively determined by second-order polynomial equation and 3D response surface plots. The biosorption mechanism was explored by characterization of the biosorbent before and after biosorption using atomic force microscopy (AFM), scanning electron microscopy, energy dispersive X-ray spectroscopy, X-ray diffraction, and Fourier transform infrared spectroscopy. The results showed that the maximum adsorption capacity of 9.6 mg/g was obtained at the initial lead ion concentration of 108.79 mg/l, pH value of 5.75, and biosorbent dosage of 9.9 g/l (fresh weight), which was close to the theoretically expected value of 9.88 mg/g. Arthrobacter sp. 25 is an ellipsoidal-shaped bacterium covered with extracellular polymeric substances. The biosorption mechanism involved physical adsorption and microprecipitation as well as ion exchange, and functional groups such as phosphoryl, hydroxyl, amino, amide, carbonyl, and phosphate groups played vital roles in adsorption. The results indicate that Arthrobacter sp. 25 may be potentially used as a biosorbent for low-concentration lead(II) removal from wastewater.

Achieving High Yield of Lactic Acid for Antimicrobial Characterization in Cephalosporin-Resistant Lactobacillus by the Co-Expression of theosphofructokinase and Glucokinase

  • Gong, Yahui;Li, Tiyuan;Li, Shiyu;Jiang, Zhenyou;Yang, Yan;Huang, Junli;Liu, Zhaobing;Sun, Hanxiao
    • Journal of Microbiology and Biotechnology
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    • v.26 no.6
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    • pp.1148-1161
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    • 2016
  • Lactobacilli are universally recognized as probiotics that are widely used in the adjuvant treatment of inflammatory diseases, such as vaginitis and enteritis. With the overuse of antibiotics in recent years, the lactobacilli in the human body are killed, which could disrupt the microecological balance in the human body and affect health adversely. In this work, cephalosporin-resistant Lactobacillus casei RL20 was obtained successfully from the feces of healthy volunteers, which possessed a stable genetic set. However, the shortage of lactic acid (72.0 g/l at 48 h) by fermentation did not meet the requirement for its use in medicine. To increase the production of lactic acid, the functional genes pfk and glk were introduced into the wild strain. A yield of 144.2 g/l lactic acid was obtained in the transgenic L. casei RL20-2 after fermentation for 48 h in 1 L of basic fermentation medium with an initial glucose concentration of 100 g/l and increasing antibacterial activity. These data suggested that L. casei RL20-2 that exhibited a high yield of lactic acid may be a potential probiotic to inhibit the spread of bacterial infectious diseases and may be used for vaginitis therapy.

A Human Fungal Pathogen Cryptococcus neoformans Expresses Three Distinct Iron Permease Homologs

  • Han, Kyunghwan;Do, Eunsoo;Jung, Won Hee
    • Journal of Microbiology and Biotechnology
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    • v.22 no.12
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    • pp.1644-1652
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    • 2012
  • Iron plays a key role in host-pathogen interactions. Microbial pathogens require iron for survival and virulence, whereas mammalian hosts sequester and withhold iron as a means of nutritional immunity. We previously identified two paralogous genes, CFT1 and CFT2, which encode homologs of a fungal iron permease, Cft1 and Cft2, respectively, in the human fungal pathogen Cryptococcus neoformans. Cft1 was shown to play a role in the high-affinity reductive iron uptake system, and was required for transferrin utilization and full virulence in mammalian hosts. However, no role of Cft2 has been suggested yet. Here, we identified the third gene, CFT3, that produces an additional fungal iron permease homolog in C. neoformans, and we also generated the cft3 mutant for functional characterization. We aimed to reveal distinct functions of Cft1, Cft2 and Cft3 by analyzing phenotypes of the mutants lacking CFT1, CFT2 and CFT3, respectively. The endogenous promoter of CFT1, CFT2 and CFT3 was replaced with the inducible GAL7 promoter in the wild-type strain or in the cft1 mutant for gain-of-function analysis. Using these strains, we were able to find that CFT2 is required for growth in low-iron conditions in the absence of CFT1 and that overexpression of CFT2 compensates for deficiency of the cft1 mutant in iron uptake and various cellular stress conditions. However, unlike CFT2, no clear phenotypic characteristic of the cft3 mutant and the strain overexpressing CFT3 was observed. Overall, our data suggested a redundant role of Cft2 in the high-affinity iron uptake and stress responses in C. neoformans.

Spectroscopic Characterization of Humic Substances Present in Landfill Leachates with Respect to Landfilling Age (매립 연령에 따른 침출수중에 존재하는 휴믹물질의 분광학적 특성 분석)

  • Kang, Kihoon;Shin, Hyun-Sang;Park, Heekyung
    • Journal of Korean Society of Environmental Engineers
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    • v.22 no.6
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    • pp.1113-1126
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    • 2000
  • Humic substances present in landfill leachates were extracted and their characteristics were analyzed with respect to landfilling age as a basic study to evaluate the effect of humic substances on the transport and transformation of other pollutants in pollution plume contaminated by landfill leachate. Element analysis of the extracted humic substances were performed. The information on the functional groups of the humic substances molecules were obtained from the spectroscopic analysis using UV-visible, FT/IR, and fluorescence spectroscopy. The structural characteristics of the humic substances were also analyzed by $^1H$ and $^{13}C$ NMR spectroscopy. From the results of the analyses it was found that the structure of the humic substances present in landfill leachates was more aliphatic than aromatic relative to other common humic substances extracted from natural soils and water. The results indicate that the degree of humification of the humic substances were relatively low. The degree of humification, however, was increased as the landfilling age was increased.

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Thermal properties and mechanical properties of dielectric materials for thermal imprint lithography

  • Kwak, Jeon-Bok;Cho, Jae-Choon;Ra, Seung-Hyun
    • Proceedings of the Korean Institute of Electrical and Electronic Material Engineers Conference
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    • 2006.06a
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    • pp.242-242
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    • 2006
  • Increasingly complex tasks are performed by computers or cellular phone, requiring more and more memory capacity as well as faster and faster processing speeds. This leads to a constant need to develop more highly integrated circuit systems. Therefore, there have been numerous studies by many engineers investigating circuit patterning. In particular, PCB including module/package substrates such as FCB (Flip Chip Board) has been developed toward being low profile, low power and multi-functionalized due to the demands on miniaturization, increasing functional density of the boards and higher performances of the electric devices. Imprint lithography have received significant attention due to an alternative technology for photolithography on such devices. The imprint technique. is one of promising candidates, especially due to the fact that the expected resolution limits are far beyond the requirements of the PCB industry in the near future. For applying imprint lithography to FCB, it is very important to control thermal properties and mechanical properties of dielectric materials. These properties are very dependent on epoxy resin, curing agent, accelerator, filler and curing degree(%) of dielectric materials. In this work, the epoxy composites filled with silica fillers and cured with various accelerators having various curing degree(%) were prepared. The characterization of the thermal and mechanical properties wasperformed by thermal mechanical analysis (TMA), thermogravimetric analysis (TGA), differential scanning calorimetry (DSC), rheometer, an universal test machine (UTM).

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Characterization of the molecular and biological properties between the equine herpesvirus type 1 immediate-early protein and the general transcription factor human TFIIB

  • Jang Hyung-Kwan
    • Korean Journal of Veterinary Service
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    • v.27 no.4
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    • pp.355-369
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    • 2004
  • The equine herpesvirus type 1 (EHV-1) immediate-early (IE) protein is a potent transactivator responsible for the activation of both early and late genes during the course of infection and is comprised of discrete functional domains that mediate its many functions. Interaction between trans activators such as the IE protein and various components of the RNA polymerase II transcription initiation machinery has been demonstrated to be critical for transactivation. In the present report, it is addressed the hypothesis that the IE protein interacts with various components of transcription machinery to mediate transactivation of target viral genes. In these studies, it is demonstrated that in vitro transcribed and translated IE protein interacts with TFIIB-agarose conjugate but not with TFIID-agarose conjugate. Additional immunoprecipitation studies using nuclear extracts derived from EHV-1 infected RK-13 cells confirmed that the IE protein interacts strongly with TFIIB, but fails to interact with TFIID. IR2, a truncated form of the IE protein lacking the potent transactivation domain and involved in the down-regulation of the IE gene, also interacted with TFIIB but not with TFIID. Studies were also performed to ascertain if particular TBP-associated factors (TAFs) could mediate IE or IR2 binding to TFIID. In vitro transcribed and translated TAF250 added to nuclear extracts generated from EHV-1 infected cells also failed to mediate an interaction between the IE protein or the IR2 protein and TFIID. This study demonstrated that the IE protein mediates transactivation of target viral genes by a mechanism that involves TFIIB. This is in contrast to mechanisms that have been proposed for both the herpes simplex virus ICP4 and VP16 protein which have been proposed to transactivate viral genes through interactions involving both TFIIB and TFIID. This study also intimates that IR2 mediate its repressive effects during the course of EHV-1 infection by a mechanism that involves sequestration of various transcription factors.

Microbial Rhodopsins: Genome-mining, Diversity, and Structure/Function

  • Jung, Kwang-Hwan;Vishwa Trivedi;Yang, Chii-Shen;Oleg A. Sineschekov;Elena N. Spudich;John L. Spudich
    • Journal of Photoscience
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    • v.9 no.3
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    • pp.45-48
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    • 2002
  • Microbial rhodopsins, photoactive 7-transmembrane helix proteins that use retinal as their chromophore, were observed initially in the Archaea and appeared to be restricted to extreme halophilic environments. Our understanding of the abundance and diversity of this family has been radically transformed by findings over the past three years. Genome sequencing of cultivated microbes as well as environmental genomics have unexpectedly revealed archaeal rhodopsin homologs in the other two domains of life as well, namely Bacteria and Eucarya. Organisms containing these homologs inhabit such diverse environments as salt flats, soil, freshwater, and surface and deep ocean waters, and they comprise a broad phylogenetic range of microbial life, including haloarchaea, proteobacteria, cyanobacteria, fungi, and algae. Analysis of the new microbial rhodopsins and their expression and structural and functional characterization reveal that they fulfill both ion transport and sensory functions in various organisms, and use a variety of signaling mechanisms. We have obtained the first crystallographic structure for a photosensory member of this family, the phototaxis receptor sensory rhodopsin II (SRII, also known as phoborhodopsin) that mediates blue-light avoidance by the haloarchaeon Natronobacterium pharaonis. The structure obtained from x-ray diffraction of 3D crystals prepared in a cubic lipid phase reveals key features responsible for its spectral tuning and its sensory function. The mechanism of SRII signaling fits a unified model for transport and signaling in this widespread family of phototransducers.

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Characterization of tryptophan residues of human coagulation factor V required for binding to phospholipid membranes (인지질막 결합에 필요한 제5혈액응고인자 트립토판잔기들의 역할규명)

  • Kim, Suhng-Wook
    • Journal of Life Science
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    • v.13 no.4
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    • pp.463-472
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    • 2003
  • Interactions between factor Va (HFVa) and membrane phosphatidylserine (PS) regulate the activity of the prothrombinase complex. I have previously shown that two solvent exposed hydrophobic residues located in the C2-domain, Trp2063 and Trp2064, are required for binding to immobilized PS and for expression of procoagulant activity on membranes containing 5% PS. In order to fully define the functional importance of these two residues I have expressed and isolated recombinant factor Va (rHFVa) W2063A/W2064A double mutant. In contrast to the native protein the two glycoforms resulting from alternative glycosylation of Asn2181 eluted as a single peak with rHFVa1 W2063A/W2064A eluting on the leading edge and rHFVa2 W2063A/W2064A eluting on the trailing edge. The double mutant rHFVa2 W2063A/W2064A expressed little or no procoagulant activity on membranes containing 1-10% mol % PS. In contrast, the procoagulant activity of this mutant was slightly greater than the native protein on membranes containing>18 mol % PS. The binding of rHFVa2 W2063A/W2064A to immobilized phospholipid vesicles was markedly reduced compared to the native protein in a surface plasmon resonance binding assay. I conclude that Trp2063 and Trp2064 are required for high affinity binding of factor Va to PS membranes and that this interaction is necessary for assembly of the prothrombinase complex on membranes containing physiological concentrations of PS.