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REFERENCE LINKING PLATFORM OF KOREA S&T JOURNALS
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Journal of Microbiology and Biotechnology
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Journal DOI :
The Korean Society for Applied Microbiology and Biotechnology
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Volume & Issues
Volume 23, Issue 12 - Dec 2013
Volume 23, Issue 11 - Nov 2013
Volume 23, Issue 10 - Oct 2013
Volume 23, Issue 9 - Sep 2013
Volume 23, Issue 8 - Aug 2013
Volume 23, Issue 7 - Jul 2013
Volume 23, Issue 6 - Jun 2013
Volume 23, Issue 5 - May 2013
Volume 23, Issue 4 - Apr 2013
Volume 23, Issue 3 - Mar 2013
Volume 23, Issue 2 - Feb 2013
Volume 23, Issue 1 - Jan 2013
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Generation and Expression in Plants of a Single-Chain Variable Fragment Antibody Against the Immunodominant Membrane Protein of Candidatus Phytoplasma Aurantifolia
Shahryari, F. ; Safarnejad, M.R. ; Shams-Bakhsh, M. ; Schillberg, S. ; Nolke, G. ;
Journal of Microbiology and Biotechnology, volume 23, issue 8, 2013, Pages 1047~1054
DOI : 10.4014/jmb.1301.01054
Witches' broom of lime is a disease caused by Candidatus Phytoplasma aurantifolia, which represents the most significant global threat to the production of lime trees (Citrus aurantifolia). Conventional disease management strategies have shown little success, and new approaches based on genetic engineering need to be considered. The expression of recombinant antibodies and fragments thereof in plant cells is a powerful approach that can be used to suppress plant pathogens. We have developed a single-chain variable fragment antibody (scFvIMP6) against the immunodominant membrane protein (IMP) of witches' broom phytoplasma and expressed it in different plant cell compartments. We isolated scFvIMP6 from a naïve scFv phage display library and expressed it in bacteria to demonstrate its binding activity against both recombinant IMP and intact phytoplasma cells. The expression of scFvIMP6 in plants was evaluated by transferring the scFvIMP6 cDNA to plant expression vectors featuring constitutive or phloem specific promoters in cassettes with or without secretion signals, therefore causing the protein to accumulate either in the cytosol or apoplast. All constructs were transiently expressed in Nicotiana benthamiana by agroinfiltration, and antibodies of the anticipated size were detected by immunoblotting. Plant-derived scFvIMP6 was purified by affinity chromatography, and specific binding to recombinant IMP was demonstrated by enzyme-linked immunosorbent assay. Our results indicate that scFvIMP6 binds with high activity and can be used for the detection of Ca. Phytoplasma aurantifolia and is also a suitable candidate for stable expression in lime trees to suppress witches' broom of lime.
Cloning and mRNA Expression Analysis of the Gene Encoding Phenylalanine Ammonia-Lyase of the Ectomycorrhizal Fungus Tricholoma matsutake
Yoon, Hyeokjun ; You, Young-Hyun ; Kim, Ye-Eun ; Kim, Young Ja ; Kong, Won-Sik ; Kim, Jong-Guk ;
Journal of Microbiology and Biotechnology, volume 23, issue 8, 2013, Pages 1055~1059
DOI : 10.4014/jmb.1303.03064
The ectomycorrhizal fungus Tricholoma matsutake grows symbiotically with Pinus densiflora. Phenylalanine ammonia-lyase (E.C. 188.8.131.52) catalyzes the conversion of L-phenylalanine to trans-cinnamic acid. The role of fungal phenylalanine ammonia-lyase, however, has not been clear until now. In this study, the gene encoding phenylalanine ammonia-lyase (PAL), which was isolated from T. matsutake, was cloned and characterized. The PAL gene (tmpal) consists of 2,160 nucleotides, coding for a polypeptide containing 719 amino acid residues. The deduced amino acid sequence of tmpal from T. matsutake shows high identity (70%) with that from Laccaria bicolor. Comparative analysis of the PAL genes among T. matsutake and other species of the class Agaricomycetes showed that both active sites and binding sites were significantly conserved among these genes. The transcriptional analysis of the PAL gene revealed a differential gene expression pattern depending on the developmental stages (mycelium, primordium, stipe, pileus, and gills) of T. matsutake. These results suggest that the PAL gene in T. matsutake plays an important role in multiple physiological functions.
Molecular Cloning and Enzymatic Characterization of Cyclomaltodextrinase from Hyperthermophilic Archaeon Thermococcus sp. CL1
Lee, Jae-Eun ; Kim, In-Hwan ; Jung, Jong-Hyun ; Seo, Dong-Ho ; Kang, Sung-Gyun ; Holden, James F. ; Cha, Jaeho ; Park, Cheon-Seok ;
Journal of Microbiology and Biotechnology, volume 23, issue 8, 2013, Pages 1060~1069
DOI : 10.4014/jmb.1302.02073
Genome organization near cyclomaltodextrinases (CDases) was analyzed and compared for four different hyperthermophilic archaea: Thermococcus, Pyrococcus, Staphylothermus, and Thermofilum. A gene (CL1_0884) encoding a putative CDase from Thermococcus sp. CL1 (tccd) was cloned and expressed in Escherichia coli. TcCD was confirmed to be highly thermostable, with optimal activity at
. The melting temperature of TcCD was determined to be
by both differential scanning calorimetry and differential scanning fluorimetry. A size-exclusion chromatography experiment showed that TcCD exists as a monomer. TcCD preferentially hydrolyzed
-CD), and at the initial stage catalyzed a ring-opening reaction by cleaving one
-1,4-glycosidic linkage of the CD ring to produce the corresponding single maltooligosaccharide. Furthermore, TcCD could hydrolyze branched CDs (G1-
-CD, and G2-
-CD) to yield significant amounts (45%, 40%, and 46%) of isomaltooligosaccharides (panose and
-maltosylmaltose) in addition to glucose and maltose. This enzyme is one of the most thermostable maltogenic amylases reported, and might be of potential value in the production of isomaltooligosaccharides in the food industry.
Effect of the Antimicrobial Peptide
-Nal-Pac-525 on the Growth of Streptococcus mutans and Its Biofilm Formation
Li, Huajun ; Cheng, Jya-Wei ; Yu, Hui-Yuan ; Xin, Yi ; Tang, Li ; Ma, Yufang ;
Journal of Microbiology and Biotechnology, volume 23, issue 8, 2013, Pages 1070~1075
DOI : 10.4014/jmb.1212.12035
Streptococcus mutans is the primary etiological agent of dental caries. The antimicrobial peptide
-Nal-Pac-525 was designed by replacing the tryptophans of the Trp-rich peptide Pac-525 with
-naphthyalanines. To assess the effect of
-Nal-Pac-525 on cariogenic bacteria, the activity of
-Nal-Pac-525 on the growth of S. mutans and its biofilm formation were examined.
-Nal-Pac-525 showed robust antimicrobial activity against S. mutans (minimum inhibitory concentration of 4
). Using scanning electron microscopy and transmission electron microscopy, it was shown that
-Nal-Pac-525 caused morphological changes and damaged the cell membrane of S. mutans.
-Nal-Pac-525 inhibited biofilm formation of S. mutans at 2
. The results of this study suggest that
-Nal-Pac-525 has great potential for clinical application as a dental caries-preventing agent.
In Vitro Screening for Compounds Derived from Traditional Chinese Medicines with Antiviral Activities Against Porcine Reproductive and Respiratory Syndrome Virus
Cheng, Jia ; Sun, Na ; Zhao, Xin ; Niu, Li ; Song, Meiqin ; Sun, Yaogui ; Jiang, Junbing ; Guo, Jianhua ; Bai, Yuansheng ; He, Junping ; Li, Hongquan ;
Journal of Microbiology and Biotechnology, volume 23, issue 8, 2013, Pages 1076~1083
DOI : 10.4014/jmb.1303.03074
Seventeen compounds derived from traditional Chinese medicines (TCMs) were tested for their antiviral activity against porcine reproductive and respiratory syndrome virus (PRRSV) in vitro. Visualization with the cytopathologic effect (CPE) assay and the 3-(4, 5-dimethyithiazol-2-yl)-2,5-diphenyltetrazolium bromide test were used to determine the 50% cytotoxic concentration (
) and 50% effective concentration (
) in cultured Marc-145 cells. Among the tested compounds, chlorogenic acid and scutellarin showed potential anti-PRRSV activity. The
and the selectivity indexes were >5.54 and 35.5, respectively. The time-of-addition and virucidal assay indicated that the anti-PRRSV activity of the two compounds could be due to their inhibiting the early stage of virus replication and/or inactivating the virus directly. The inhibition of the virus attachment was not observed in the adsorption inhibition assay. The inhibition ratios of chlorogenic acid and scutellarin were, respectively, 90.8% and 61.1% at the maximum non-cytotoxic concentrations. The results have provided a basis for further exploration of their antiviral properties and mechanisms in vivo. We believe that the chlorogenic acid and scutellarin have a great potential to be developed as new anti-PRRSV drugs for clinical application.
Statistical Optimization of Medium Components for Milk-Clotting Enzyme Production by Bacillus amyloliquefaciens D4 Using Wheat Bran-an Agro-Industry Waste
Zhang, Weibing ; He, Xiaoling ; Liu, Hongna ; Guo, Huiyuan ; Ren, Fazheng ; Gao, Weidong ; Wen, Pengcheng ;
Journal of Microbiology and Biotechnology, volume 23, issue 8, 2013, Pages 1084~1091
DOI : 10.4014/jmb.1212.12043
In this paper, two statistical methods were applied to optimize medium components to improve the production of the milk-clotting enzyme by Bacillus amyloliquefaciens D4. First, wheat bran juice, skim milk powder, and
were shown to have significant effects on D4 enzyme production using the Plackett-Burman experimental design. Subsequently, an optimal medium was obtained using the Box-Behnken method, which consisted of 3.31 g/l of skim milk powder, 5.0 g/l of sucrose, 0.1 g/l of
, 0.1 g/l of
, 0.1 g/l of
, 0.1 g/l of
, 1.52 g/l of
, and 172.45 g/l of wheat bran juice. With this optimal medium, the milk-clotting enzyme production was remarkably enhanced. The milk-clotting enzyme activity reached 3,326.7 SU/ml after incubation of 48 h, which was 1.76-fold higher than that of the basic medium, showing that the Plackett-Burman design and Box-Behnken response surface method are effective to optimize medium components, and B. amyloliquefaciens D4 possessed a high rennet-producing capacity in the optimal medium.
Acetone-Butanol-Ethanol (ABE) Production in Fermentation of Enzymatically Hydrolyzed Cassava Flour by Clostridium beijerinckii BA101 and Solvent Separation
Lepiz-Aguilar, Leonardo ; Rodriguez-Rodriguez, Carlos E. ; Arias, Maria Laura ; Lutz, Giselle ;
Journal of Microbiology and Biotechnology, volume 23, issue 8, 2013, Pages 1092~1098
DOI : 10.4014/jmb.1301.01021
Cassava constitutes an abundant substrate in tropical regions. The production of butanol in ABE fermentation by Clostridium beijerinckii BA101 using cassava flour (CF) was scaled-up to bioreactor level (5 L). Optimized fermentation conditions were applied; that is,
, 60 g/l CF, and enzymatic pretreatment of the substrate. The batch fermentation profile presented an acidogenic phase for the first 24 h and a solventogenic phase afterwards. An average of 37.01 g/l ABE was produced after 83 h, with a productivity of 0.446 g/l/h. Butanol production was 25.71 g/l with a productivity of 0.310 g/l/h, high or similar to analogous batch processes described for other substrates. Solvent separation by different combinations of fractioned and azeotropic distillation and liquid-liquid separation were assessed to evaluate energetic and economic costs in downstream processing. Results suggest that the use of cassava as a substrate in ABE fermentation could be a cost-effective way of producing butanol in tropical regions.
Efficiency and Midgut Histopathological Effect of the Newly Isolated Bacillus thuringiensis KS
-Endotoxins on the Emergent Pest Tuta absoluta
Jamoussi, Kais ; Sellami, Sameh ; Nasfi, Zina ; Krichen-Makni, Saloua ; Tounsi, Slim ;
Journal of Microbiology and Biotechnology, volume 23, issue 8, 2013, Pages 1099~1106
DOI : 10.4014/jmb.1301.01035
Tuta absoluta (Povolny, 1994) is a devastating moth to the Solanaceae plants. It is a challenging pest to control, especially on tomatoes. In this work, we studied the entomopathogenic activity of the Cry-forming
-endotoxins produced by Bacillus thuringiensis strain KS and B. thuringiensis kurstaki reference strain HD1 against T. absoluta. These strains carried the cry2, cry1Ab, cry1Aa/cry1Ac, and cry1I genes, and KS also carried a cry1C gene. The
-endotoxins of KS were approximately twofold more toxic against the third instar larvae than those of HD1, as they showed lower 50% and 90% lethal concentrations (0.80 and 2.70
-endotoxins/tomato leaf)) compared with those of HD1 (1.70 and 4.50
) (p < 0.05). Additionally, the larvae protease extract showed at least six caseinolytic activities, which activated the KS and HD1
-endotoxins, yielding the active toxins of about 65 kDa and the protease-resistant core of about 58 kDa. Moreover, the histopathological effects of KS and HD1
-endotoxins on the larvae midgut consisted of an apical columnar cell vacuolization, microvillus damage, and epithelial cell disruption. These results showed that the KS strain could be a candidate for T. absoluta control.
Intermolecular Interaction Between Cry2Aa and Cyt1Aa and Its Effect on Larvicidal Activity Against Culex quinquefasciatus
Bideshi, Dennis K. ; Waldrop, Greer ; Fernandez-Luna, Maria Teresa ; Diaz-Mendoza, Mercedes ; Wirth, Margaret C. ; Johnson, Jeffrey J. ; Park, Hyun-Woo ; Federici, Brian A. ;
Journal of Microbiology and Biotechnology, volume 23, issue 8, 2013, Pages 1107~1115
DOI : 10.4014/jmb.1301.01062
The Cyt1Aa protein of Bacillus thuringiensis susbp. israelensis elaborates demonstrable toxicity to mosquito larvae, but more importantly, it enhances the larvicidal activity of this species Cry proteins (Cry11Aa, Cry4Aa, and Cry4Ba) and delays the phenotypic expression of resistance to these that has evolved in Culex quinquefasciatus. It is also known that Cyt1Aa, which is highly lipophilic, synergizes Cry11Aa by functioning as a surrogate membrane-bound receptor for the latter protein. Little is known, however, about whether Cyt1Aa can interact similarly with other Cry proteins not primarily mosquitocidal; for example, Cry2Aa, which is active against lepidopteran larvae, but essentially inactive or has very low toxicity to mosquito larvae. Here we demonstrate by ligand binding and enzyme-linked immunosorbent assays that Cyt1Aa and Cry2Aa form intermolecular complexes in vitro, and in addition show that Cyt1Aa facilitates binding of Cry2Aa throughout the midgut of C. quinquefasciatus larvae. As Cry2Aa and Cry11Aa share structural similarity in domain II, the interaction between Cyt1Aa and Cry2Aa could be a result of a similar mechanism previously proposed for Cry11Aa and Cyt1Aa. Finally, despite the observed interaction between Cry2Aa and Cyt1Aa, only a 2-fold enhancement in toxicity resulted against C. quinquefasciatus. Regardless, our results suggest that Cry2Aa could be a useful component of mosquitocidal endotoxin complements being developed for recombinant strains of B. thuringiensis subsp. israelensis and B. sphaericus aimed at improving the efficacy of commercial products and avoiding resistance.
Engineering the Cellular Protein Secretory Pathway for Enhancement of Recombinant Tissue Plasminogen Activator Expression in Chinese Hamster Ovary Cells: Effects of CERT and XBP1s Genes
Rahimpour, Azam ; Vaziri, Behrouz ; Moazzami, Reza ; Nematollahi, Leila ; Barkhordari, Farzaneh ; Kokabee, Leila ; Adeli, Ahmad ; Mahboudi, Fereidoun ;
Journal of Microbiology and Biotechnology, volume 23, issue 8, 2013, Pages 1116~1122
DOI : 10.4014/jmb.1302.02035
Cell line development is the most critical and also the most time-consuming step in the production of recombinant therapeutic proteins. In this regard, a variety of vector and cell engineering strategies have been developed for generating high-producing mammalian cells; however, the cell line engineering approach seems to show various results on different recombinant protein producer cells. In order to improve the secretory capacity of a recombinant tissue plasminogen activator (t-PA)-producing Chinese hamster ovary (CHO) cell line, we developed cell line engineering approaches based on the ceramide transfer protein (CERT) and X-box binding protein 1 (XBP1) genes. For this purpose, CERT S132A, a mutant form of CERT that is resistant to phosphorylation, and XBP1s were overexpressed in a recombinant t-PA-producing CHO cell line. Overexpression of CERT S132A increased the specific productivity of t-PA-producing CHO cells up to 35%. In contrast, the heterologous expression of XBP1s did not affect the t-PA expression rate. Our results suggest that CERT-S132A-based secretion engineering could be an effective strategy for enhancing recombinant t-PA production in CHO cells.
Cr(VI) Resistance and Removal by Indigenous Bacteria Isolated from Chromium-Contaminated Soil
Long, Dongyan ; Tang, Xianjin ; Cai, Kuan ; Chen, Guangcun ; Shen, Chaofeng ; Shi, Jiyan ; Chen, Linggui ; Chen, Yingxu ;
Journal of Microbiology and Biotechnology, volume 23, issue 8, 2013, Pages 1123~1132
DOI : 10.4014/jmb.1301.01004
The removal of toxic Cr(VI) by microorganisms is a promising approach for Cr(VI) pollution remediation. In the present study, four indigenous bacteria, named LY1, LY2, LY6, and LY7, were isolated from Cr(VI)-contaminated soil. Among the four Cr(VI)-resistant isolates, strain LY6 displayed the highest Cr(VI)-removing ability, with 100 mg/l Cr(VI) being completely removed within 144 h. It could effectively remove Cr(VI) over a wide pH range from 5.5 to 9.5, with the optimal pH of 8.5. The amount of Cr(VI) removed increased with initial Cr(VI) concentration. Data from the time-course analysis of Cr(VI) removal by strain LY6 followed first-order kinetics. Based on the 16S rRNA gene sequence, strain LY6 was identified as Pseudochrobactrum asaccharolyticum, a species that had never been reported for Cr(VI) removal before. Transmission electron microscopy and energy dispersive X-ray spectroscopy analysis further confirmed that strain LY6 could accumulate chromium within the cell while conducting Cr(VI) removal. The results suggested that the indigenous bacterial strain LY6 would be a new candidate for potential application in Cr(VI) pollution bioremediation.
Enzymatic Hydrolysis of Gelatin Layers of X-Ray Films and Release of Silver Particles Using Keratinolytic Serine Proteases from Purpureocillium lilacinum LPS # 876
Cavello, Ivana A. ; Hours, Roque A. ; Cavalitto, Sebastian F. ;
Journal of Microbiology and Biotechnology, volume 23, issue 8, 2013, Pages 1133~1139
DOI : 10.4014/jmb.1302.02038
Enzymatic decomposition of gelatin layers on used X-ray films and repeated utilization of the enzyme for potential application in silver recovery were investigated using keratinolytic serine proteases from Purpureocillium lilacinum LPS # 876. At pH 9.0, the enzymatic reaction was enhanced by the increase of enzyme concentration or by the increase of the temperature up to
. Under the conditions of 6.9 U/ml,
, and pH 9.0, hydrolysis of the gelatin layers and the resulting release of silver particles were achieved within 6 min. The protective effect of polyols against thermal denaturation was investigated. The presence of glycerol and propylene glycol increased enzyme stability. When the reusability of the enzyme for gelatin hydrolysis was tested, it could be seen that it could be effectively reused for more cycles when glycerol was added, compared with the enzyme without protective agents. The results of these repeated treatments suggested that a continuous process of recycling silver from used X-ray is feasible. Keeping in mind that recycling is (at the present time) needed and imperative, it can be remarked that, in this research, three wastes were successfully used: hair waste in order to produce serine proteases; glycerol in order to enhance enzyme thermal stability; and used X-ray films in order to recover silver and PET films.
Production of Acetate from Carbon Dioxide in Bioelectrochemical Systems Based on Autotrophic Mixed Culture
Su, Min ; Jiang, Yong ; Li, Daping ;
Journal of Microbiology and Biotechnology, volume 23, issue 8, 2013, Pages 1140~1146
DOI : 10.4014/jmb.1304.04039
Bioelectrochemical systems (BESs) have been suggested as a new technology for wastewater treatment while accomplishing energy and chemical generation. This study describes the performance of BESs based on mixed culture that are capable of reducing carbon dioxide to acetate. The cathode potential was a critical factor that affected the performance of the BESs. The rate of acetate production increased as the electrode potential became more negative, from 0.38 mM
(-900 mV vs. Ag/AgCl) to 2.35 mM
(-1,100 mV), while the electron recovery efficiency of carbon dioxide reduction to acetate increased from 53.6% to 89.5%. The microbial population was dominated by relatives of Acetobacterium woodii when a methanogenic inhibitor was added to the BESs initially.
Biocontrol of Pectobacterium carotovorum subsp. carotovorum Using Bacteriophage PP1
Lim, Jeong-A ; Jee, Samnyu ; Lee, Dong Hwan ; Roh, Eunjung ; Jung, Kyusuk ; Oh, Changsik ; Heu, Sunggi ;
Journal of Microbiology and Biotechnology, volume 23, issue 8, 2013, Pages 1147~1153
DOI : 10.4014/jmb.1304.04001
Pectobacterium carotovorum subsp. carotovorum (formerly Erwinia carotovora subsp. carotovora) is a plant pathogen that causes soft rot and stem rot diseases in several crops, including Chinese cabbage, potato, and tomato. To control this bacterium, we isolated a bacteriophage, PP1, with lytic activity against P. carotovorum subsp. carotovorum. Transmission electron microscopy revealed that the PP1 phage belongs to the Podoviridae family of the order Caudovirales, which exhibit icosahedral heads and short non-contractile tails. PP1 phage showed high specificity for P. carotovorum subsp. carotovorum, and several bacteria belonging to different species and phyla were resistant to PP1. This phage showed rapid and strong lytic activity against its host bacteria in liquid medium and was stable over a broad range of pH values. Disease caused by P. carotovorum subsp. carotovorum was significantly reduced by PP1 treatment. Overall, PP1 bacteriophage effectively controls P. carotovorum subsp. carotovorum.
Detection of Human Cytomegalovirus UL97 D605E Mutation in Korean Stem Cell Transplantation Recipients and Donors
Lee, Gyu-Cheol ; Choi, Su-Mi ; Lee, Chan Hee ; Lee, Dong-Gun ; Choi, Jung-Hyun ; Yoo, Jin-Hong ;
Journal of Microbiology and Biotechnology, volume 23, issue 8, 2013, Pages 1154~1158
DOI : 10.4014/jmb.1303.03094
Ganciclovir resistance of human cytomegalovirus is associated with mutations in the viral UL97 gene and poses severe problems for immunocompromised patients. In this study, PCR-based restriction fragment length polymorphism and sequencing analyses detected the UL97 D605E mutation in all five clinical isolates from patients with ganciclovir-resistant human cytomegalovirus infection during prolonged ganciclovir therapy, whereas the M460V mutation was only present in 1 of 5 isolates. On the other hand, the detection rates of the D605E mutation in the stored available DNA samples from the donor and allogeneic stem cell transplantation recipients were 66.7% and 93.7%, respectively, suggesting that the presence of D605E mutation was not associated with the ganciclovir exposure. Although the D605E mutation may not be related to ganciclovir resistance, we suggest that this mutation could be an important molecular marker of human cytomegalovirus evolution in East Asian countries. Moreover, the restriction fragment length polymorphism method using the restriction enzyme HaeIII, which is generally used to detect the UL97 A591V mutation, could also detect the D605E mutation and may therefore be a useful tool for future research on the investigation of UL97 gene mutations.
Orientia tsutsugamushi Infection Induces
T Cell Activation via Human Dendritic Cell Activity
Chu, Hyuk ; Park, Sung-Moo ; Cheon, In Su ; Park, Mi-Yeoun ; Shim, Byoung-Shik ; Gil, Byoung-Cheol ; Jeung, Woon Hee ; Hwang, Kyu-Jam ; Song, Ki-Duk ; Hong, Kee-Jong ; Song, Manki ; Jeong, Hang-Jin ; Han, Seung Hyun ; Yun, Cheol-Heui ;
Journal of Microbiology and Biotechnology, volume 23, issue 8, 2013, Pages 1159~1166
DOI : 10.4014/jmb.1303.03019
Orientia tsutsugamushi, a gram-negative bacterium, causes severe acute febrile illness in humans. Despite this danger, the route of infection, infectivity, and protective mechanisms of the host's immune response to O. tsutsugamushi are unclear. Dendritic cells (DCs) are one of the most important cell types in bridging the innate and adaptive immune responses. In this study, we observed that O. tsutsugamushi infects and replicates in monocyte-derived DCs (MODCs). During infection and replication, the expressions of the cytokines IL-12 and TNF-
, as well as the co-stimulatory molecules CD80, CD83, CD86, and CD40, were increased in MODCs. When O. tsutsugamushi-treated MODCs were co-cultured with autologous
T cells, they enhanced production of IFN-
, a major Th1 cytokine. Collectively, our results show that O. tsutsugamushi can replicate in MODCs and can simultaneously induce MODC maturation and increase proinflammatory cytokine levels in MODCs that subsequently activate
Analysis of Transcriptional Profiles to Discover Biomarker Candidates in Mycobacterium avium subsp. paratuberculosis-Infected Macrophages, RAW 264.7
Cha, Seung Bin ; Yoo, Anna ; Park, Hong Tae ; Sung, Kyoung Yong ; Shin, Min Kyoung ; Yoo, Han Sang ;
Journal of Microbiology and Biotechnology, volume 23, issue 8, 2013, Pages 1167~1175
DOI : 10.4014/jmb.1302.02021
Paratuberculosis (PTB) or Johne's disease is one of the most serious chronic debilitating diseases of ruminants worldwide that is caused by Mycobacterium avium subsp. paratuberculosis (MAP). MAP is a slow-growing bacterium that has very long latent periods, resulting in difficulties in diagnosing and controlling the disease, especially regarding the diagnosis of fecal shedders of MAP without any clinical signs. Based on this situation, attempts were made to identify biomarkers that show early responses to MAP infection in a macrophage cell line, RAW 264.7. In response to the infection with the bacterium, a lot of genes were turned on and/or off in the cells. Of the altered genes, three different categories were identified based on the time-dependent gene expression patterns. Those genes were considered as possible candidates for biomarkers of MAP infection after confirmation by quantitative RT-PCR analysis. To the best of our knowledge, this is the first attempt at discovering the host transcriptomic biomarkers of PTB, although further investigation will be required to determine whether these biomarker candidates are associated within the natural host.
Rapid Establishment of CHO Cell Lines Producing the Anti-Hepatocyte Growth Factor Antibody SFN68
Song, Seong-Won ; Lee, Song-Jae ; Kim, Chang-Young ; Han, Byungryeul ; Oh, Jong-Won ;
Journal of Microbiology and Biotechnology, volume 23, issue 8, 2013, Pages 1176~1184
DOI : 10.4014/jmb.1305.05056
Anti-hepatocyte growth factor (anti-HGF) monoclonal antibodies (mAbs) are potential therapeutics against various cancers. Screening for high-producer clones is a time-consuming and complex process and is a major hurdle in the development of therapeutic mAbs. Here, we describe an efficient approach that allows the selection of high-producer Chinese hamster ovary (CHO) cell lines producing the novel anti-HGF mAb SFN68, which was generated previously by immunizing HGF bound to its receptor c-Met. We selected an SFN68-producing parental cell line via transfection of the dihydrofolate reductase-deficient CHO cell line DG44, which was preadapted to serum-free suspension culture, with an SFN68-expression vector. Subsequent gene amplification via multiple passages of the parental cell line in a methotrexate-containing medium over 4 weeks, followed by clonal isolation, enabled us to isolate two cell lines, 2F7 and 2H4, with 3-fold higher specific productivity. We also screened 72 different media formulated with diverse feed and basal media to develop a suboptimized medium. In the established suboptimized medium, the highest anti-HGF mAb yields of the 2F7 and 2H4 clones were 842 and 861 mg/l, respectively, which were about 10.5-fold higher than that of the parental cell line in a non-optimized basal medium. The selected CHO cell lines secreting high titers of SFN68 would be useful for the production of sufficient amounts of antibodies for efficacy evaluation in preclinical and early clinical studies.