• Journal of Microbiology and Biotechnology
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• v.25 no.7
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• pp.1015-1025
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• 2015

The development of diverse polymyxin derivatives is needed to solve the toxicity and resistance problems of polymyxins. However, no platform has generated polymyxin derivatives by genetically engineering a polymyxin synthetase, which is a nonribosomal peptide synthetase. In this study, we present a two-step approach for the construction of engineered polymyxin synthetases by substituting the adenylation (A) domains of polymyxin A synthetase, which is encoded by the pmxABCDE gene cluster of Paenibacillus polymyxa E681. First, the seventh L-threonine-specific A-domain region in pmxA was substituted with the L-leucine-specific A-domain region obtained from P. polymyxa ATCC21830 to make polymyxin E synthetase, and then the sixth D-leucine-specific A-domain region (A_6-D-Leu-domain) was substituted with the D-phenylalanine-specific A-domain region (A_6-D-Phe-domain) obtained from P. polymyxa F4 to make polymyxin B synthetase. This step was performed in Escherichia coli on a pmxA-containing fosmid, using the lambda Red recombination system and the sacB gene as a counter-selectable marker. Next, the modified pmxA gene was fused to pmxBCDE on the chromosome of Bacillus subtilis BSK4dA, and the resulting recombinant strains BSK4-PB and BSK4-PE were confirmed to produce polymyxins B and E, respectively. We also succeeded in constructing the B. subtilis BSK4-PP strain, which produces polymyxin P, by singly substituting the A_6-D-Leu-domain with the A_6-D-Phe-domain. This is the first report in which polymyxin derivatives were generated by genetically engineering polymyxin synthetases. The two recombinant B. subtilis strains will be useful for improving the commercial production of polymyxins B and E, and they will facilitate the generation of novel polymyxin derivatives.

• JSTS:Journal of Semiconductor Technology and Science
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• v.13 no.5
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• pp.511-515
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• 2013

A Monte Carlo (MC) simulation study has been done in order to investigate the effects of line-edge-roughness (LER) induced by either 1P1E (single-patterning and single-etching) or 2P2E (double-patterning and double-etching) on fully-depleted silicon-on-insulator (FDSOI) tri-gate metal-oxide-semiconductor field-effect transistors (MOSFETs). Three parameters for characterizing the LER profile [i.e., root-mean square deviation (${\sigma}$), correlation length (${\zeta}$), and fractal dimension (D)] are extracted from the image-processed scanning electron microscopy (SEM) image for each photolithography method. It is experimentally verified that two parameters (i.e., ${\sigma}$ and D) are almost the same in each case, but the correlation length in the 2P2E case is longer than that in the 1P1E case. The 2P2E-LER-induced $V_TH$ variation in FDSOI tri-gate MOSFETs is smaller than the 1P1E-LER-induced $V_TH$ variation. The total random variation in $V_TH$, however, is very dependent on the other major random variation sources, such as random dopant fluctuation (RDF) and work-function variation (WFV).

• Proceedings of the Korean Society of Crop Science Conference
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• 1988.02a
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• pp.100-101
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• 1988

• Journal of Marine Life Science
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• v.3 no.2
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• pp.81-86
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• 2018

The purpose of this study is to determine the effects of vibration on primary (e.g. plasma cortisol), secondary (e.g. plasma glucose, aspartate aminotransferase (AST), alanine aminotransferase (ALT), Na⁺, K⁺ and Cl^- and tertiary (e.g. mortality) stress responses in cultured eel, Anguilla japonica. For this purpose, three groups (including one control group and two stress groups) were set up. The control group was made exposed to vibration corresponding to 48 decibel (dB, V) (produced using electric vibrators) for 15 minutes per hour every day, and the two stress groups was made exposed to vibration corresponding to 58 and 68 dB (V) (produced using the same electric vibrators), equally, for 15 minutes per hour every day. Blood was sampled at day 0 (before starting vibration stress tests, BS), and days 1, 3, 5, 7, 9 and 11 (after starting vibration stress tests). As a result, plasma cortisol showed trend to continuously rise by consecutive stress from 4.1±0.1 ng/ml in BS. In 48 dB group (control), cortisol showed the highest level with 7.6±0.9 ng/ml after 7 days (p<0.05), but at 9 and 11 days was not significantly compared with BS level. In 58 dB group, the cortisol showed the highest level with 43.1±4.8 ng/ml after 1st day. Cortisol of 68 dB group increased significantly during the experimental period (14.4±2.3~32.0±5.7 ng/ml) (p<0.05). In 58, and 68 dB groups during the experimental period differed significantly compared to 48 group (p<0.05). Glucose in 48 dB were increased from 42.0 ±5.7 (BS) to 52.5±2.1 (1 day), the level was not significantly from 1 to 11 days. Glucose in 58 and 68 dB groups was increased significantly than BS during experimental period (p<0.05). K⁺ in 68 dB increased significantly (p<0.05) from 2.3±0.2 mE/ql (BS) to 3.3±0.5 mE/ql at 5 days. In 48 and 58 dB groups during the experimental period differed significantly (p<0.05). Na⁺ and Cl^- levels were not differed significantly during the experimental period. AST and ALT in 58 and 68 dB groups showed trend to continuously rise by consecutive stress. At 7 and 9 days in AST, between 48, 58 and 68 dB groups differed significantly (p<0.05). In 48, 58 and 68 dB groups at 1 day, blood hematocrit increased significantly higher than BS. The 11 days after vibration stress, the mortality in 48, 58 and 68 dB groups was 1.1, 5.1 and 5.8%, respectively. The present results have shown that A. japonica exhibited ''typical'' physiological responses when exposed to chronic vibration stress. These data suggested that chronic vibration stress caused substantial stress in the fish; especially the persisting elevated plasma AST and ALT levels observed would be expected to adverse effect. In conclusion, chronic vibration stress could greatly affects the hematological characteristics in A. japonica.

• Journal of Plant Biology
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• v.34 no.4
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• pp.331-339
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• 1991

To elucidate the regulatory mechanism of virE operon from vir regions (virA, virB, virC, virD, virG, virE) of pTiA6 which have been known to be essential for efficient crown gall tumorigenesis in plants, the activity of the truncated virE, promoter was analyzed. pSM358cd, a recombinant plasmid in which virE :: Tn3-HoHo1 (Tn3-promoterless lacZ) was cloned into SalI site of pVK102, was digested with SalI, and virE :: Tn3-HoHo1 was seperated from pVK102. To construct the truncted virE recombinant plasmids (pJS031, pJS051, pJS102, pJS201, pJS301), 5'-end of vireE promoter was deleted with BAL31 and cloned into pVK102 and then transferred into a. tumefaciens A348(pTiA6). According to the activity of the truncated virE promoter in recombinant plasmids, they were classified into two groups, pJS031, pJS051, pJS101 and pJS201 belong to a functional group and pJS301 is a non-functional. The size of deleted nucleotides of pJS201 and pJS301 seemed to be about 130 nucleotides and about 250 nucleotides from 5'-end of virE promoter, respectively. Hence it was thought that the essential site of the virE promoter was located between about 130th nucleotide and 250th nucleotide from 5'-end of the virE promoter.

• The Journal of the Korean bone and joint tumor society
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• v.14 no.2
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• pp.106-118
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• 2008

Purpose: Disturbed cell cycle regulatory proteins are key events underlying the development and/or progression of human malignancies. The aim of this study is to evaluate the protein expression status involved in G1/S cell cycle in human soft tissue sarcoma. Materials and Methods: We simultaneously evaluated the expression of Cyclin D1, Cyclin E, CDK4, CDK2, p16, p27, Rb, E2F1, p53 and Ki-67 by immunohistochemistry in 43 cases of soft tissue sarcoma Results: The Cyclin D1, Cyclin E, CDK4, CDK2, E2F1, and p53 were expressed in 25 (58.1%), 18 (41.9%), 13 (30.2%), 33 (76.7%), 20 (46.5%), and 18 cases (41.9%). The p16, p27, and Rb expressions were decreased in 26 (60.5%), 22 (51.2%) and 19 cases (44.2%). All of the cases showed alterations of more than one out of the above proteins. The increased Cyclin E expression and Ki-67 labeling index (LI) were significantly associated with histologic grade. The Cyclin E and E2F-1 expressions were increased in relapsed cases and the CDK4 expression was increased in cases of metastasis. Conclusion: Alterations of G1/S cell cycle regulatory proteins may play an important role in the tumoriogensis of soft tissue sarcomas. Our results suggest that increased expressions of Cyclin E, E2F1, and CDK4 were associated with tumor relapse or metastasis and could be considered as parameters of prognosis of soft tissue sarcoma.

• Proceedings of the Korea Air Pollution Research Association Conference
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• 1998.10a
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• pp.223-224
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• 1998

• Journal of Preventive Medicine and Public Health
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• v.32 no.1
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• pp.88-94
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• 1999

Objectives. In order to gain a better understanding of the mechanism of DMF toxicity, recent studies have focused on hepatic drug metabolizing enzymes. In this study, we investigated the effects of DMF on the induction of P450 and the activities of other related enzymes in rat liver microsomes. Methods. DMF was administered to male Sprague Daweley rats by intraperitoneal injection at 0(control), 450(D1), 900(D2), 1,800(D3) mg DMF/kg body weight in olive oil once a day for three days. Hepatic P450 was measured by method of Omura and Sato. We evaluated selective assays for the three drug metabolizing cytochrome P450 isoenzymes 1A1, 2B1 and 2E1. Results. The content of microsomal protein, P450 and b5 were tended to be decreased in DMF treated group, but they were not statistically significant. The activity of NADPH-cytochrome P450 reductase was significantly increased dose dependently(p<0.01), but the activity of NADH-b5 reductase was decreased in the treated group(p<0.01). The activities of PROD and EROD were not significant between control and treated group. The activities of pNPH in the DMF treated groups were higher than that of the control group(p<0.01). When Western immunoblottings were carried out utilizing three monoclonal antibodies which were specific against P4501A1/1, P4502B1/2 and P4502E1, the strong density band corresponding to P4502E1 was observed with the microsomes obtained from the rats treated with DMF. But there were no significant increased in the P4501A1/2 and P4502B1/2 band densities in immunoblotting. Conclusions. These result suggested that P4502E1 was inducible by DMF and P4502E1 isozyme might be responsible for the hydroxylation of DMF to HMMF.

• Korean Journal of Microbiology
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• v.28 no.2
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• pp.169-173
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• 1990

For the overproduction of L-phenylalanine using Escherichia coli, the authors constructed various recombinant plasmids including pMW 10, pMW 11 and pMW 12. The $aroF{FR}$ and $pheA^{FR}$ genes for the production of L-phenylalanine were isolated from Escherichia coli MWEC 101-5 strains. The productivity and atability of Escherichia coli regulatory mutants containing recombinant plasmids were investigated to evaluate the efficiency of the $aroF^{FR}$ and $pheA^{FR}$ genes. The MWEC 101-5/pMW 11 strain produced 24.3g/l of L-phenylalanine while its stability was 73.8 percent. The specific activity of prephenate dehydratase in the MWEC 101-5/pMW 11 strain increased by 26-fold compared with that of Escherichia coli K-12.

• Journal of Microbiology and Biotechnology
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• v.2 no.4
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• pp.243-247
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• 1992

The thr operon of Escherichia coli TF427, an $\alpha$-amino-$\beta$-hydroxyvaleric acid (AHV)-resistant threonine overproducer, was cloned in a pBluescriptII $KS^＋$ plasmid by complementation of E. coli mutants. All clones contained a common 8.8 kb HindIII-generated DNA fragment and complemented the thrA, thrB, and thrC mutants by showing that these clones contained the whole thr operon. This thr operon was subcloned in the plasmid vectors pBR322, pUC18, and pECCG117, an E. coli/Corynebacterium glutamicum shuttle vector, to form recombinant plasmids pBTF11, pUTF25 and pGTF18, respectively. The subcloned thr operon was shown to be present in a 6.0 kb insert. A transformant of E. coli TF125 with pBTF11 showed an 8~11 fold higher aspartokinase I activity, and 15~20 fold higher L-threonine production than TF125, an AHV-sensitive methionine auxotroph. Also, it was found that the aspartokinase I activity of E. coli TF125 harboring pBTF11 was not inhibited by threonine and its synthesis was not repressed by threonine plus isoleucine.