Browsing by Author "Tseng, Ching-Ping"

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A mutant strain of microalga Chlorella sp for the carbon dioxide capture from biogas
(2012-01-01)
Kao, Chien-Ya
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Chiu, Sheng-Yi
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Huang, Tzu-Ting
;
Dai, Le
;
Wang, Guan-Hua
To upgrade biogas produced from the anaerobic digestion of swine wastewater, an outdoor photobioreactor was established in this study. A mutant strain of microalga Chlorella sp. MM-2 was firstly isolated by ethyl methane sulfonate-induced random mutagenesis. The Chlorella sp. MM-2 grew in the presence of gas containing H2S < 100 ppm, and the growth capacity of the microalgal culture aerated with 80% CH, was similar to 70% that of the control culture (0% CH4). In the field study, CO2 capture efficiency of the Chlorella cultures, at a biomass concentration of 1.2 g L-1, from the desulfurized biogas (-20% CO2, 70% CH4 and H2S < 100 ppm) was approximate 70% on cloudy days and 80% on sunny days. CH4 concentration in the effluent biogas from the Chlorella cultures was increased to approximate 84% on cloudy days and 87% on sunny days from its original 70%. The established outdoor photobioreactor system using a gas cycle-switching operation could be used as a CO2 capture model for biogas upgrading. Crown Copyright (C) 2011 Published by Elsevier Ltd. All rights reserved.
Analysis of microbial diversity and optimal conditions for enhanced biogas production from swine waste anaerobic digestion
(2013-09-01)
Chung, Ying-Chien
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Chang, Yu-Cheng
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Chen, Yu-Pei
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Lin, Wei-Chih
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Lin, Hsiao-Hsien
Swine wastewater pretreated by solid-liquid separation was optimized for biogas production and water purification. Dynamic diversity of the bacterial community in the anaerobic plug flow reactor was investigated under various temperatures and hydraulic retention times (HRT). Results of batch experiments indicated that under optimal operating conditions, biogas production, methane production rate, and chemical oxygen demand (COD) removal efficiencies increased up to 26%, 52%, and 44%, respectively. In the continuous experiments, optimal operating conditions for the plug flow operator were found to be HRT of 8 d and temperature of 35 degrees C, under which relatively high CH4 concentration (67%), CH4 production rate (4.6 l/d), and total COD removal (79%) were achieved. A minimum of 12 bands was observed in the denaturing gradient gel electrophoresis profile. Phylogenetic analysis revealed the predominance of phyla Firmicutes, Proteobacteria, and Euryarchaeota in the anaerobic plug-flow reactor. However, the occurrence and prevalence of specific bacterial species varied with HRT and temperature. This study establishes the optimal operating parameters for the anaerobic plug-flow digestion of swine wastewater, which can be employed in a full-scale reactor to maximize biogas production and to purify water quality. (C) 2013 AIP Publishing LLC.
Application of aurintricarboxylic acid for the adherence of mouse P19 neurons and primary hippocampal neurons to noncoated surface in serum-free culture
(2012-11-01)
Lee, Tsung-Yih
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Chen, Wen-Shin
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Huang, Yung-An
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Liu, Ting-Wei
;
Hwang, Eric
Dissociated primary neuron culture has been the most widely used model systems for neuroscience research. Most of these primary neurons are cultured on adhesion matrix-coated surface to provide a proper environment for cell anchorage under serum-free conditions. In this study, we provide an alternative technique to promote the adhesions of these neurons using aurintricarboxylic acid (ATA), a nonpeptide compound, without surface manipulations. We first demonstrated that ATA could promote Chinese hamster ovary cell attachment and proliferation in serum-free medium in a dosage-dependent manner. We later showed that ATA significantly enhanced the attachment of the retinoic acid differentiated P19 mouse embryonal carcinoma (P19) neurons, with an optimal concentration around 30 mu g/mL. A similar result was seen in primary hippocampal neurons, with an optimal ATA concentration around 15 mu g/mL. Further morphological assessments revealed that the average neurite length and neuronal polarization were almost identical to that obtained using a conventional method with poly-L-lysine surface. The advantages of using the ATA treatment technique for immunochemical analysis are discussed. (C) 2012 American Institute of Chemical Engineers Biotechnol. Prog., 2012
Aurintricarboxylic acid inhibits influenza virus neuraminidase
(2009-02-01)
Hung, Hui-Chen
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Tseng, Ching-Ping
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Yang, Jinn-Moon
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Ju, Yi-Wei
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Tseng, Sung-Nain
There is a continuing threat that the highly pathogenic avian influenza virus will cause future influenza pandemics. In this study, we screened a library of compounds that are biologically active and structurally diverse for inhibitory activity against influenza neuraminidase (NA). We found that aurintricarboxylic acid (ATA) is a potent inhibitor of NA activity of both group-1 and group-2 influenza viruses with IC(50)S (effective concentration to inhibit NA activity by 50%) values at low micromolar concentrations. ATA was equally potent in inhibiting the NA activity derived from wild-type NA and its H274Y mutant which renders NA resistance to inhibition by oseltamivir. Although ATA is structurally distinct from sialic acid, molecular modeling experiments suggested that ATA binds to NA at the enzyme's substrate binding site. These results indicate that ATA may be a good starting material for the design of a novel class of NA inhibitors for the treatment influenza viruses. (C) 2008 Elsevier B.V. All rights reserved.
Biofiltration of trimethylamine, dimethylamine, and methylamine by immobilized Paracoccus sp CP2 and Arthrobacter sp CP1
(2008-05-01)
Ho, Kuo-Ling
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Chung, Ying-Chien
;
Lin, Yueh-Hsien
;
Tseng, Ching-Ping
;
生物科技學系
A biofilter using granular activated carbon with immobilized Paracoccus sp. CP2 was applied to the elimination of 10-250 ppm of trimethylamine (TMA), dimethylamine (DMA), and methylamine (MA). The results indicated that the system effectively treated MA (>93%), DMA (>90%), and TMA (>85%) under high loading conditions, and the maximum degradation rates were 1.4, 1.2, and 0.9 g-N kg(-1) GAC d(-1). Among the three different amines treated, TMA was the most difficult to degrade and resulted in ammonia accumulation. Further study on TMA removal showed that the optimal pH was near neutral (6.0-8.0). The supply of high glucose (>0.1%) inhibited TMA removal, maybe due to substrate competition. However, complete TMA degradation was achieved under the co-immobilization of Paracoccus sp. CP2 and Arthrobacter sp. CP1 (similar to 96%). Metabolite analysis results demonstrated that the metabolite NH(4)(+) concentrations decreased by a relatively small 27% while the metabolite NO(2)(-) apparently increased by heterotrophic nitrification of Arthrobacter sp. CP1 in the co-immobilization biofilter. (C) 2008 Elsevier Ltd. All rights reserved.
Biomass and lipid production of a novel microalga, Chlorellaceae sp P5, through heterotrophic and swine wastewater cultivation
(2018-05-01)
Chiang, Yin-Ling
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Chen, Yu-Pei
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Yu, Ming-Chen
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Hsieh, Sung-Yuan
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Hwang, Ing-Er
A potential novel genus of microalgae was isolated from swine wastewater. According to a phylogenetic analysis of 18S rRNA and internal transcribed spacer (ITS) sequences, as well as the ITS2 secondary structure, this strain was designated as Chlorellaceae sp. P5. Acclimatization to various temperatures, pH conditions, and salinities demonstrated that this strain can grow at temperatures of 20 degrees C-40 degrees C, in an acidity range of pH 4-7, and with salinity as high as 3% NaCl. To optimize biomass production, response surface methodology was used which included variables associated with carbon and nitrogen sources. In this study, flask cultivation yielded 1.66 g l(-1) d(-1) of biomass under 15.19 g l(-1) glucose and 8.26 mM ammonium bicarbonate. The fatty acid composition suggested that the lipid from Chlorellaceae sp. P5 can be typically used for biodiesel production, with a low degree of unsaturated fatty acids. Moreover, a high content of the first-limiting amino acid (lysine) and 9,12-Octadecadienoic acid (linoleic acid, C18:2) for nutritional supplements was also observed. With respect to swine wastewater treatment, the isolated strain showed an excellent removal rate of ammonia and phosphate over 97.3 mg l(-1) of NH3-N and 20.3 mg of l(-1) PO43- in the sterilized and none-sterilized wastewater. The biomass and lipid derived from Chlorellaceae sp. P5 can be attained at 0.49 g l(-1) d(-1) and 0.13 g l(-1) d(-1) by swine wastewater cultivation with 5% CO2 supply, respectively. According to the various characteristic analyses, Chlorellaceae sp. P5 has great potential for different industrial developments such as biodiesel and wastewater treatment. Published by AIP Publishing.
Characterization of MRT, a new non-LTR retrotransposon in Monascus spp
(2007-10-01)
Chen, Yi-Pei
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Tseng, Ching-Ping
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Liaw, Li-Ling
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Wang, Chun-Lin
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Yuan, Gwo-Fang
A new non-LTR retrotransposon, named MRT, was discovered in the filamentous fungus Monascus pilosus BCRC38072. The entire nucleotide sequence of the MRT element was 5.5-kb long, including two open reading frames. These two ORFs showed homologies to gag-like and pol-like gene products, and an A-rich sequence at the 3' end of pol-like gene. ORF1 encoded a protein of 5 17 amino acids and contained a cysteine-rich zinc finger motif ORF2 encoded a protein of 1181 amino acids and contained apurinic/apyrimidinic endonuclease (APE), reverse transcriptase (RT), RNaseH domains, and a CCHC motif. The phylogenetic analyses demonstrated that the MRT element should be classified into the Tad1 clade. The results of Southern hybridizations showed that MRT elements were distributed within M pilosus, M ruber, M sanguineus, and M barkeri. In addition, the species of Monascus can be grouped by the presence or absence of MRT elements in the hybridization pattern according to phylogenctic subgroups established with the partial beta-tubulin gene.
Cloning and characterization of monacolin K biosynthetic gene cluster from Monascus pilosus
(2008-07-23)
Chen, Yi-Pei
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Tseng, Ching-Ping
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Liaw, Li-Ling
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Wang, Chun-Lin
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Chen, I-Ching
Monacolin K is a secondary metabolite synthesized by polyketide synthases (PKS) from Monascus, and it has the same structure as lovastatin, which is mainly produced by Aspergillus terreus. In the present study, a bacterial artificial chromosome (BAC) clone, mps01, was screened from the BAC library constructed from Monascus pilosus BCRC38072 genomic DNA. The putative monacolin K biosynthetic gene cluster was found within a 42 kb region in the mps01 clone. The deduced amino acid sequences encoded by the nine genes designated as mokA-mokl, which share over 54% similarity with the lovastatin biosynthetic gene cluster in A. terreus, were assumed to be involved in monacolin K biosynthesis. A gene disruption construct designed to replace the central part of mokA, a polyketide synthase gene, in wild-type M. pilosus BCRC38072 with a hygromycin B resistance gene through homologous recombination, resulted in a mokA-disrupted strain. The disruptant did not produce monacolin K, indicating that mokA encoded the PKS responsible for monacolin K biosynthesis in M. pilosus BCRC38072.
Continuous deodorization and bacterial community analysis of a biofilter treating nitrogen-containing gases from swine waste storage pits
(2008-05-01)
Ho, Kuo-Ling
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Chung, Ying-Chien
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Tseng, Ching-Ping
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生物科技學系
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Department of Biological Science and Technology
A biofilter inoculated with Arthrobacter sp. was applied to the simultaneous elimination of trimethylamine (TMA) and ammonia (NH3) from the exhaust air of swine waste storage pits. The results showed that the biofilter achieved average removal efficiencies of 96.8 +/- 2.5% and 97.2 +/- 2.3% for TMA and NH3, respectively. A near-neutral pH (7.3-7.4) was maintained due to the accumulation of acid metabolites and the adsorption of alkaline NH3. Low moisture demand, low pressure drop and high biofilm stability in the system were other advantages. After long-term operation, the bacterial community structure showed that at least twenty-five bands were explicitly detected by a denaturing gradient gel electrophoresis (DGGE) method. However, the inoculated Arthrobacter sp. still maintained a dominant population (> 50%). Paracoccus denitrificans' presence in the biofilter could play an important role in oxidizing NH3 and reducing nitrite by heterotrophic nitrification and anaerobic denitrification. (c) 2007 Published by Elsevier Ltd.
CRP represses the CRISPR/Cas system in Escherichia coli: evidence that endogenous CRISPR spacers impede phage P1 replication
(2014-06-01)
Yang, Chi-Dung
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Chen, Yen-Hua
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Huang, Hsi-Yuan
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Huang, Hsien-Da
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Tseng, Ching-Ping
The CRISPR/Cas system is an important aspect in bacterial immunology. The anti-phage activity of the CRISPR system has been established using synthetic CRISPR spacers, but in vivo studies of endogenous CRISPR spacers are relatively scarce. Here, we showed that bacteriophage P1 titre in Escherichia coli decreased in the glucose-containing medium compared with that in the absence of glucose. This glucose effect of E. coli against phage P1 infection disappeared in cse3 deletion mutants. The effect on the susceptibility to phage P1 was associated with cAMP receptor protein (CRP)-mediated repression of cas genes transcription and crRNA maturation. Analysis of the regulatory element in the cse1 promoter region revealed a novel CRP binding site, which overlapped with a LeuO binding site. Furthermore, the limited sequence identity between endogenous spacers and the phage P1 genome was necessary and sufficient for CRISPR-mediated repression of phage P1 replication. Trans-expression of the third and seventh spacers in the CRISPR I region or third and sixth spacers in the CRISPR II region effectively reduced phage P1 titres in the CRISPR deletion mutants. These results demonstrate a novel regulatory mechanism for cas repression by CRP and provide evidence that endogenous spacers can repress phage P1 replication in E. coli.
Disparate mesenchyme-lineage tendencies in mesenchymal stem cells from human bone marrow and umbilical cord blood
(2006-03-01)
Chang, Yu-Jen
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Shih, Daniel Tzu-Bi
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Tseng, Ching-Ping
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Hsieh, Tzu-Bou
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Lee, Don-Ching
Bone marrow and umbilical cord blood are reported to be the main sources of mesenchymal stem cells (MSCs), which have been proposed for many clinical applications. This study evaluated and quantitated the differentiation potential of bone marrow-derived MSCs (bmMSCs) and cord blood-derived MSCs (cbMSCs) by in vitro induction. Results indicated that cbMSCs had a significantly stronger osteogenic potential but lower capacity for adipogenic differentiation than bmMSCs. Leptin, an important regulator of mesenchymal differentiation, has a significantly stronger effect of promoting osteogenesis and inhibiting adipogenesis in bmMSCs than in cbMSCs. Moreover, Cbfa1 mRNA expression in bmMSCs and cbMSCs was affected to different degrees by leptin during osteogenesis. In contrast, leptin reduced PPAR gamma 2 mRNA expression to the same level during adipogenesis in both types of MSCs. These results demonstrate the disparate capacities of MSCs from bone marrow and cord blood and suggest that they be used differently in experimental and therapeutic studies. In addition, the disparate differentiation tendencies of MSCs from different sources should be considered in further applications.
Elimination of high concentration hydrogen sulfide and biogas purification by chemical-biological process
(2013-08-01)
Ho, Kuo-Ling
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Lin, Wei-Chih
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Chung, Ying-Chien
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Chen, Yu-Pei
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Tseng, Ching-Ping
A chemical-biological process was performed to remove a high concentration of H2S in biogas. The high iron concentration tolerance (20 g L-1) of Acidithiobacillus ferrooxidans CP9 provided sufficient ferric iron level for stable and efficient H2S elimination. A laboratory-scale apparatus was setup for a 45 d operation to analyze the optimal conditions. The results reveal that the H2S removal efficiency reached 98% for 1500 ppm H2S. The optimal ferric iron concentration was kept between 9 and 11 g L-1 with a cell density of 10(8) CFU g(-1) granular activated carbon and a loading of 15 g S m(-3) h(-1). In pilot-scale studies for biogas purification, the average inlet H2S concentration was 1645 ppm with a removal efficiency of up to 97% for a 311 d operation and an inlet loading 40.8 g S m(-3) h(-1). When 0.1% glucose was added, the cell density increased twofold under the loading of 65.1 g S m(-3) h(-1) with an H2S removal efficiency still above 96%. The analysis results of the distribution of microorganisms in the biological reactor by DGGE show that microorganism populations of 96.7% and 62.7% were identical to the original strain at day 200 and day 311, respectively. These results clearly demonstrate that ferric iron reduction by H2S and ferrous iron oxidation by A. ferrooxidans CP9 are feasible processes for the removal of H2S from biogas. (C) 2013 Elsevier Ltd. All rights reserved.
Exploring the Distribution of Citrinin Biosynthesis Related Genes among Monascus Species
(2008-12-24)
Chen, Yi-Pei
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Tseng, Ching-Ping
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Chien, I-Ling
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Wang, Wei-Yi
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Liaw, Li-Ling
Citrinin, a hepato-nephrotoxic compound to humans, can be produced by the food fermentation microorganisms Monascus spp. In this study, we investigated the distribution of mycotoxin citrinin biosynthesis genes in 18 Monascus strains. The results show that the acyl-transferase and keto-synthase domains of the pksCT gene encoding citrinin polyketide synthase were found in Monascus purpureus, Monascus kaoliang, and Monascus sanguineus. Furthermore, the ctnA gene, a major activator for citrinin biosynthesis, was found in M. purpureus and U kaoliang, but was absent in M. sanguineus. The orf3 gene encoding oxygenase, located between pksCT and ctnA, was also present in M. purpureus and M. kaoliang. The pksCT gene was highly conserved in M. purpureus, U kaoliang, and M. sanguineus, while the ctnA and orf3 genes were shown to be highly homologous in M. purpureus and M. kaoliang. In contrast, the PCR and Southern blot analyses suggest that pksCT, ctnA, and orf3 were absent or significantly different in Monascus pilosus, Monascus ruber, Monascus barkeri, Monascus floridanus, Monascus lunisporas, and Monascus pallens. A citrinin-producing phenotype was detected only in M. purpureus and M., kaoliang using high performance liquid chromatography (HPLC). These results clearly indicate that the highly conserved citrinin gene cluster in M. purpureus and U kaoliang carry out citrinin biosynthesis. In addition, according to the phylogenetic subgroups established with the beta-tubulin gene, the citrinin gene cluster can group the species of Monascus.
GeNOSA: inferring and experimentally supporting quantitative gene regulatory networks in prokaryotes
(2015-07-01)
Chen, Yi-Hsiung
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Yang, Chi-Dung
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Tseng, Ching-Ping
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Huang, Hsien-Da
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Ho, Shinn-Ying
Motivation: The establishment of quantitative gene regulatory networks (qGRNs) through existing network component analysis (NCA) approaches suffers from shortcomings such as usage limitations of problem constraints and the instability of inferred qGRNs. The proposed GeNOSA framework uses a global optimization algorithm (OptNCA) to cope with the stringent limitations of NCA approaches in large-scale qGRNs. Results: OptNCA performs well against existing NCA-derived algorithms in terms of utilization of connectivity information and reconstruction accuracy of inferred GRNs using synthetic and real Escherichia coli datasets. For comparisons with other non-NCA-derived algorithms, OptNCA without using known qualitative regulations is also evaluated in terms of qualitative assessments using a synthetic Saccharomyces cerevisiae dataset of the DREAM3 challenges. We successfully demonstrate GeNOSA in several applications including deducing condition-dependent regulations, establishing high-consensus qGRNs and validating a sub-network experimentally for dose-response and time-course microarray data, and discovering and experimentally confirming a novel regulation of CRP on AscG.
Glucocorticoids may compromise the effect of gefitinib in non-small cell lung cancer
(2016-12-27)
Wang, Hsian-Yu
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Chang, Yu-Ling
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Cheng, Chun-Chun
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Chao, Min-Wu
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Lin, Su-I
The epidermal growth factor receptor (EGFR)-targeting tyrosine kinase inhibitors (TKIs) have shown remarkable benefits in non-small cell lung cancer (NSCLC) patients with drug-sensitive mutations in the EGFR gene. Responsive patients are usually continuously prescribed with TKIs until disease progression. Glucocorticoids (GCs) are potent homeostasis maintaining drugs and are frequently used in cancer patients to alleviate discomforts caused by anti-cancer therapies. Several previous studies reported that concomitant use of GCs may compromise the efficacy of chemo-therapeutics in patients with solid tumors. Little is known in the concomitant use of target therapy with GCs in treating NSCLC. In this study, we hypothesized that concomitant use of GCs in EGFR-TKI therapy may be detrimental and addressed this issue using cell cultures and xenograft studies followed by a retrospective population study based on data from the Taiwan national health insurance system. In cell cultures and xenograft studies, GCs were shown to unequally compromise the anti-cancer efficacy of TKIs in both PC9 and NCI-H1975 NSCLC cells models. In the retrospective population study, patients with similar disease status that were co-medicated with GCs had a significantly higher risk of disease progression.
High glucose enhances cAMP level and extracellular signal-regulated kinase phosphorylation in Chinese hamster ovary cell: Usage of Br-cAMP in foreign protein beta-galactosidase expression
(2017-07-01)
Lin, Hsiao-Hsien
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Lee, Tsung-Yih
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Liu, Ting-Wei
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Tseng, Ching-Ping
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生物科技學系
Glucose is a carbon source for Chinese hamster ovary (CHO) cell growth, while low growth rate is considered to enhance the production of recombinant proteins. The present study reveals that glucose concentrations higher than 1 g/L reduce the growth rate and substantially increase in cAMP (similar to 300%) at a high glucose concentration (10 g/L). High glucose also enhances the phosphorylation of extracellular signal-regulated kinase (ERK) and p27(kiP) by Western blot analysis. To determine whether the phosphorylation of ERK is involved in the mechanism, a cyclic-AMP dependent protein kinase A (PICA) inhibitor (H-8) or MEK (MAPKK) inhibitor (PD98059) was added to block ERK phosphorylation. We show that both the high glucose-induced ERIC phosphorylation and growth rate return to baseline levels. These results suggest that the cAMP/PKA and MAP signaling pathways are involved in the above mentioned mechanism. Interestingly, the direct addition of 8-bromo-cAMP (Br-cAMP), a membrane-permeable cAMP analog, can mimic the similar effects produced by high glucose. Subsequently Br-cAMP could induce beta-galactosidase (beta-Gal) recombinant protein expression by 1.6-fold. Furthermore, Br-cAMP can additionally enhance the (beta-Gal production (from 2.8- to 4.5 fold) when CHO cells were stimulated with glycerol, thymidine, dimethyl sulfoxide, pentanoic acid, or sodium butyrate. Thus, Br-cAMP may be used as an alternative agent in promoting foreign protein expression for CHO cells. (C) 2017, The Society for Biotechnology, Japan. All rights reserved.
High Growth Rate Downregulates fumA mRNA Transcription but Is Dramatically Compensated by Its mRNA Stability in Escherichia coli
(2012-05-01)
Lin, Hsiao-Hsien
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Lin, Ching-Hsueh
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Hwang, Shiaw-Min
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Tseng, Ching-Ping
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生物科技學系
Little is known about the association among the transcription, post-transcription, and protein production of the fumA gene. This study demonstrates that increasing growth rate (k) from 0.24/h to 0.96/h causes a marked eightfold reduction in fumA transcription as assessed using the beta-galactosidase activity from fumA promoter fused with a lacZ reporter. It was further confirmed using Northern blot analysis. Most interestingly, the FumA protein levels remained unchanged over the growth rate, as indicated by Western blot analysis. Therefore, whether the reduced fumA mRNA expression under the high growth rate can be overcome by increasing the stability of the fumA mRNA was tested. The half-life of fumA mRNA was established to significantly increase by fivefold when the growth rate was increased to 0.96/h. This finding suggests that the cells could turn down the expression of fumA mRNA because of increased stability of its mRNA under the high growth rate. This notion indicates that mRNA stability plays an essential role in maintaining a critical cellular level of a given protein when the mRNA transcript is downregulated by a metabolic event.
Identification of the mokH Gene Encoding Transcription Factor for the Upregulation of Monacolin K Biosynthesis in Monascus pilosus
(2010-01-13)
Chen, Yi-Pei
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Yuan, Gwo-Fang
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Hsieh, Sung-Yuan
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Lin, Yu-Shan
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Wang, Wei-Yi
Monacolin K is a secondary metabolite synthesized by polyketide synthases (PKS) from Monascus. The monacolin K biosynthetic gene cluster, mokA-mokI, has been characterized in Monascus pilosus. The mokH gene encoding Zn(II)2Cys6 binuclear DNA binding protein is assumed to be an activator for monacolin K production. In this study, the mokH gene was cloned and driven by the glyceraldehyde-3-phosphate dehydrogenase (gpd) promoter for overexpression in M. pilosus. The transformants containing an extra copy of the mokH gene were obtained and verified by PCR and Southern hybridization. The transcripts of mokH in the transformants were expressed significantly higher than those of the wild-type strain. The transformants were stably inherited through the next generation, as determined by observation of the enhanced green fluorescent protein (EGFP). The transformant T-mokH1 also showed a 1.7-fold higher production of monacolin K than the wild-type strain in a time course analysis. Analysis of the RT-PCR products demonstrated that the monacolin K biosynthetic genes in the transformant were expressed to a greater extent than those in the wildtype strain. These results indicated that mokH upregulated the transcription of monacolin K biosynthetic genes and increased monacolin K production.
Inhibition of enterovirus 71 replication and the viral 3D polymerase by aurintricarboxylic acid
(2010-04-01)
Hung, Hui-Chen
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Chen, Tzu-Chun
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Fang, Ming-Yu
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Yen, Kuei-Jung
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Shih, Shin-Ru
Enterovirus 71 (EV71) causes serious diseases in humans. The aim of this study was to examine the effects of aurintricarboxylic acid (ATA) on EV71 replication and to explore the underlying mechanism. To measure the activity of ATA in inhibiting the cytopathic effect (CPE) of EV71, a cell-based neutralization (inhibition of virus-induced CPE) assay was performed. The effect of ATA was further confirmed using plaque reduction and viral yield reduction assays. A time of addition assay was performed to identify the mechanisms of ATA's anti-EV71 activity. We examined the effects of ATA on the following key steps involved in virus replication: (i) translation of the internal ribosomal entry site (IRES)-mediated viral polyprotein; (ii) the proteolytic activity of viral proteases 2A and/or 3C; and (iii) the viral 3D RNA-dependent RNA polymerase (RdRp) activity. In this study, ATA was found to be a potent inhibitor of the replication of EV71. In the antiviral neutralization assay, ATA exhibited inhibitory activity against EV71 (TW/4643/98) and EV71 (TW/2231/98). Plaque assay further demonstrated that ATA inhibited EV71 replication with an EC(50) (effective concentration at which 50% of plaques were removed) of 2.9 mu M. Studies on the mechanism of action revealed that ATA targets the early stage of the viral life cycle after viral entry. ATA was able to inhibit the RdRp activity of EV71, while neither the IRES-mediated translation of viral polyprotein nor the viral 3C protease activity was affected. Overall, the findings in this study suggest that ATA is able to effectively inhibit EV71 replication through interfering with the viral 3D polymerase.
Inhibitory Effect of Excessive Glucose on Its Biochemical Pathway and the Growth of Chinese Hamster Ovary (CHO) Cells
(2015-01-02)
Lee, Tsung-Yih
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Lin, Hsiao-Hsien
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Chen, Chien-Lung
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Hwang, Shiaw-Min
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Tseng, Ching-Ping
In this study, we found the specific growth rate of CHO cells being reduced when glucose increased from 1 to 10g/L. Under high glucose, we show a 41% increase in lactate dehydrogenase activity consistent with a 70% rate increase in lactate production. The pyruvate kinase activity was significantly increased. However, the activities of citrate synthase, isocitrate dehydrogenase, and malate dehydrogenase involved in the TCA cycle were attenuated. We propose that pyruvate produced from glycolysis is utilized in a lesser extent over the TCA cycle under high-glucose conditions. Thus, the pyruvate is largely converted to lactate, which is toxic to the cells.