• 농업과학연구
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• 제44권1호
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• pp.30-39
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• 2017

This study was performed to produce succinic acid from biomass by developing mutants of Cellulomonas flavigena in which the succinate dehydrogenase gene (sdh) is deleted. For development of succinate producing mutants, the upstream and downstream regions of sdh gene from C. flavigena and antibiotic resistance gene (neo, bla) were inserted into pKC1139, and the recombinant plasmids were transformed into Escherichia coli ET12567/pUZ8002 which is a donor strain for conjugation. C. flavigena was conjugated with the transformed E. coli ET12567/pUZ8002 to induce the deletion of sdh in chromosome of this bacteria by double-crossover recombination. Two mutants (C. flavigena H-1 and H-2), in which sdh gene was deleted in the chromosome, were constructed and confirmed by PCR. To estimate the production of succinic acid by the two mutants when the culture broth was fermented with biomass such as CMC, xylan, locust gum, and rapeseed straw; the culture broth was analyzed by HPLC analysis. The succinic acid in the culture broth was not detected as a fermentation products of all biomass. One of the reasons for this may be the conversion of succinic acid to fumaric acid by sdh genes (Cfla_1014 - Cfla_1017 or Cfla_1916 - Cfla_1918) which remained in the chromosomal DNA of C. flavigena H-1 and H-2. The other reason could be the conversion of succinyl-CoA to other metabolites by enzymes related to the bypass pathway of TCA cycle.

• 대한의생명과학회지
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• 제15권1호
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• pp.37-45
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• 2009

Insect cell cultures have become important tools in the production of biological substances for use in a variety of research, human and veterinary medicine, and pest control applications. These applications often require the introduction of foreign DNA into the cells and have generally used methods originally developed for use with human and other mammalian cell cultures. While these methods can be successfully employed, they are often less efficient with insect cells and frequently involve complex procedures or require specialized equipment. Even when they do work, they may require substantial modification because of differences in the culture medium or growth patterns of insect cells. In this study, We have optimized transfection conditions of Sf9 cell line using insect expression vector pIZT/V5-His which expresses green fluorescent protein effectively. Human stem cell factor (hSCF) is a glycoprotein that plays a key role in hematopoiesis acting both as a positive and negative regulator, often in synergy with other cytokines. It also plays a key role in mast cell development, gametogenesis, and melanogenesis. It can exist in membrane-bound form and in proteolytically released soluble form. As determined by an enzyme-linked immunosorbent assay performed, hSCF level in supernatant averaged 995ng/ml. The human hSCF was partially purified by immunoaffinity chromatography and analyzed with sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoblotting. The results show that the hSCF has N-linked carbohydrate and corresponds to the soluble form, at or about 223 amino acids in length. The findings suggest functional importance for soluble hSCF in cells.

• 미생물학회지
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• 제25권1호
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• pp.40-45
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• 1987

B Brevibacterium divaricatum FERM 5948 균주로부터 Bdi I RIM 체계에 속하는 BdiI methylase 유천자를 클로닝하여 발현을 조사하였다. Bdi I methylase 유전자의 클로닝을 위해 pBR 322의 EcoRI, BamHl, Sal I 3 군데의 클로닝 site를 이 용했고 1 차 형질전환후 나온 플라스미드를 BdiI으로 자른 뒤 ligation 시키지 않고 형질전환시키는 방법을 이용하였다. 유전 자을 가지는 행질전환체의 선별은 Bdi I methylase에 의해 수정된 채조합 플라스미드는 BdiI 제한효소에 방호된다는 것에 기 초하여 선별하였는데 5.6kb의 EcoRI insert DNA를 가지는 pBDIM 116이 Bdil methylase 유전자플 가지는 것으로 판명 되었다. pBDIM 11&을 가지는 숙주셰포에서 추출한 추출용액에는 S-adenosylmethionine이 있으면 BdiI의 인지부위인 A ATCGAT에만 특정한 methylase 활성이 측정되였다. 11개의 제한효소를 이용하이 제한효소지도를 작성하였고, BdiI r restriction -modification 체계에 관해서 도 논의하였다.

• Journal of Microbiology
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• 제40권2호
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• pp.156-160
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• 2002

The whole gal/lae operon genes of Lactococcus lactis ssp. lactis 7962 were reported as follows: galA-galM-galK-galT-lacA -lacZ-galE. The galK gene encoding a galactokinase involved in one of the Leloir pathways for galactose metabolism was found to be 1,197 bp in length and encodes a protein of 43,822 Da calculated molecular mass. The deduced amino acid sequence showed over 50% homology with GaIK proteins from several other lactic acid bacteria. The galK gene was expressed in E. coli and the product was identified as a 43 kDa protein which corresponds to the estimated size from the DNA sequence. The galactokinase activity of recombinant 5. coli was about 8 times greater against that of the host strain and more than 3 times higher than the induced L. lactis 7962.

• The Plant Pathology Journal
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• 제31권4호
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• pp.402-413
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• 2015

Yellow rust (stripe rust), caused by Puccinia striiformis f. sp. tritici, is one of the most destructive foliar diseases of wheat in Egypt and worldwide. In order to identify wheat genotypes resistant to yellow rust and develop molecular markers associated with the resistance, fifty F8 recombinant inbred lines (RILs) derived from a cross between resistant and susceptible bread wheat landraces were obtained. Artificial infection of Puccinia striiformis was performed under greenhouse conditions during two growing seasons and relative resistance index (RRI) was calculated. Two Egyptian bread wheat cultivars i.e. Giza-168 (resistant) and Sakha-69 (susceptible) were also evaluated. RRI values of two-year trial showed that 10 RILs responded with RRI value >6 <9 with an average of 7.29, which exceeded the Egyptian bread wheat cultivar Giza-168 (5.58). Thirty three RILs were included among the acceptable range having RRI value >2 <6. However, only 7 RILs showed RRI value <2. Five RILs expressed hypersensitive type of resistance (R) against the pathogen and showed the lowest Average Coefficient of Infection (ACI). Bulked segregant analysis (BSA) with eight simple sequence repeat (SSR), eight sequence-related amplified polymorphism (SRAP) and sixteen random amplified polymorphic DNA (RAPD) markers revealed that three SSR, three SRAP and six RAPD markers were found to be associated with the resistance to yellow rust. However, further molecular analyses would be performed to confirm markers associated with the resistance and suitable for marker-assisted selection. Resistant RILs identified in the study could be efficiently used to improve the resistance to yellow rust in wheat.

• 동의생리병리학회지
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• 제17권4호
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• pp.1037-1049
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• 2003

Numerous lines of evidence indicate that some of the neurotoxicity associated with Alzheimer's disease (AD) is due to proteolytic fragments of the amyloid precursor protein (APP). Most research has focused on the amyloid 6 (M). However, the possible role of other cleaved products of APP is less clear. Lately It has been reported that a recombinant carboxy-terminal 105 amino acid fragment (CT105) of APP induced strong nonselective inward currents in Xenopus oocyte. In a brain with Alzheimer's disease (AD), to investigate the roles of carboxyl-terminal fragment (CT105) of amyloid precursor protein (APP) in apoptosis processes possibly linked to neurodegeneration associated with AD, we examined the effects of the CT of APP with 105 amino acid residues (CT105) on the alteration of apoptosis triggers in neubroblastoma cells. We have investigated whether Radix Polygalae and Rhizoma Acori Graminei mixture extract (RP+RAG) inhibits CT105-induced apoptosis of neuroblastoma cells. We found that RP+RAG inhibits CT105-induced apoptosis in SK-N-SH cells. Treatment of the cells with RP+RAG inhibited CT105-induced DNA fragmentation and Tunel assay of nuclear chromatin and inhibited the caspase-3 expression in SK-N-SH cells. As the result of this study, In RP+RAG group, the apoptosis in the nervous system is inhibited, the repair against the degerneration of neuroblastoma cells by CT105 expression is promoted. These results indicate that RP+RAG possess strong inhibitory effect of apoptosis in the nervous system and repair effect against the degeneration of neuroblastoma cells by CT105 expression

• Biomolecules & Therapeutics
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• 제2권4호
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• pp.336-342
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• 1994

The general pharmacological properties of EPO were investigated in various animals administering intravenously and in vitro system. The results were as follows. 1. Central nervous system: EPO at doses of 70, 700, 7000 U/kg showed no effect In mice on general behavior, on strychnine- and pentetrazol-induced convulsion and on acetic acid-induced writhing syndrome. The hexobarbital-induced sleeping time in mice was slightly reduced by EPO at a dose of 7000 U/kg but did not change at doses of 70, 700 U/kg. The body temperature in rats was slightly decreased by EPO at doses of 700, 7,000 U/kg but the change was in normal physiological range. 2. Respiratory and cardiovascular system: EPO showed no effect on respiratory rate, blood pressure, heart rate, femoral blood flow, and electrocardiogram in anesthetized dogs at doses of 70, 700, 7000 U/kg. 3. Smooth muscle: EPO at concentrations of 70, 700 U/ml had no effect on the contractile response of isolated guinea pig ileum to histamine and acetylcholine. 4. Water and electrolytes excretion: EPO at dose above 700 U/kg increased urine volume in rats but did not affect the concentrations of $Na^{+},\;K^{+},\;Cl^{-}$ in urine. 5. Gastrointestinal system: EPO(70, 700, 7000 U/kg) had no effect on the intestinal charcoal meal propulsion 6. Blood coagulation system: The administration of EPO(70, 700, 7000 U/kg) had no effect on the plasma prothrombin time(PT) and activated partial thromboplastin time(APTT) in mice. Platelet aggregation induced by ADP and collagen was not influenced by EPO(70 U/ml, 700 U/ml). The overall results obtained indicated that EPO exerts almost no serious pharmacological effect even at a 100-fold clinical dose(7000 U/kg).

• Molecules and Cells
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• 제39권7호
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• pp.550-556
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• 2016

During meiosis, exchange of DNA segments occurs between paired homologous chromosomes in order to produce recombinant chromosomes, helping to increase genetic diversity within a species. This genetic exchange process is tightly controlled by the eukaryotic RecA homologs Rad51 and Dmc1, which are involved in strand exchange of meiotic recombination, with Rad51 participating specifically in mitotic recombination. Meiotic recombination requires an interaction between homologous chromosomes to repair programmed double-strand breaks (DSBs). In this study, we investigated the budding yeast meiosis-specific proteins Hop2 and Sae3, which function in the Dmc1-dependent pathway. This pathway mediates the homology searching and strand invasion processes. Mek1 kinase participates in switching meiotic recombination from sister bias to homolog bias after DSB formation. In the absence of Hop2 and Sae3, DSBs were produced normally, but showed defects in the DSB-to-single-end invasion transition mediated by Dmc1 and auxiliary factors, and mutant strains failed to complete proper chromosome segregation. However, in the absence of Mek1 kinase activity, Rad51-dependent recombination progressed via sister bias in the $hop2{\Delta}$ or $sae3{\Delta}$ mutants, even in the presence of Dmc1. Thus, Hop2 and Sae3 actively modulate Dmc1-dependent recombination, effectively progressing homolog bias, a process requiring Mek1 kinase activation.

• Food Science and Biotechnology
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• 제17권6호
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• pp.1221-1227
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• 2008

The application of the cellulase gene (celA) as a selection marker of food-grade integration system was investigated in Lactobacillus (Lb.) casei, Lactococcus lactis, and Leuconostoc (Leu.) mesenteroides. The 6.0-kb vector pOC13 containing celA from Clostridium thermocellum with an integrase gene and a phage attachment site originating from bacteriophage A2 was used for site-specific recombination into chromosomal DNA of lactic acid bacteria (LAB). pOC13 was also equipped with a broad host range plus replication origin from the lactococcal plasmid pWV01, and a controllable promoter of nisA ($P_{nisA}$) for the production of foreign proteins. pOC13 was integrated successfully into Lb. casei EM116, and pOC13 integrants were easily detectable by the formation of halo zone on plates containing cellulose. Recombinant Lb. casei EM 116::pOC13 maintained these traits in the absence of selection pressure during 100 generations. pOC13 was integrated into the chromosome of L. lactis and Leu. mesenteroides, and celA acted as an efficient selection marker. These results show that celA can be used as a food-grade selection marker, and that the new integrative vector could be used for the production of foreign proteins in LAB.

• Mass Spectrometry Letters
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• 제5권3호
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• pp.84-88
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• 2014

Licoricidin isolated from Glycyrrhiza uralensis is known to have anticancer, anti-nephritic, anti-Helicobacter pylori, and antibacterial effects. In this study, a cocktail probe assay and liquid chromatography-tandem mass spectrometry (LC-MS/MS) were used to investigate the modulating effect of licoricidin on cytochrome P450 (CYP) enzymes in human liver microsomes. When licoricidin was incubated at $0-25{\mu}m$ with CYP probes for 60 min at $37^{\circ}C$, it showed potent inhibitory effects on CYP2B6-catalyzed bupropion hydroxylation and CYP2C9-catalyzed diclofenac 4'-hydroxylation with half maximal inhibitory concentration ($IC_{50}$) values of 3.4 and $4.0{\mu}m$, respectively. The inhibition mode of licoricidin was revealed as competitive, dose-dependent, and non-time-dependent, and following the pattern of Lineweaver-Burk plots. The inhibitory effect of licoricidin has been confirmed in human recombinant cDNA-expressed CYP2B6 and 2C9 with $IC_{50}$ values of 4.5 and $0.73{\mu}m$, respectively. In conclusion, this study has shown the potent inhibitory effect of licoricidin on CYP2B6 and CYP2C9 activity could be important for predicting potential herb-drug interactions with substrates that mainly undergo CYP2B- and CYP2C9-mediated metabolism.