• 대한수의학회지
• /
• 제48권3호
• /
• pp.243-250
• /
• 2008

A calmodulin-dihydrofolate reductase (DHFR) sandwich fusion protein was generated by insertion of calmodulin into the $\beta$-bulge region of DHFR to observe the effects of structurally constraining the calmodulin structure. The calcium binding properties of the sandwich protein were almost identical to calmodulin. Similar to calmodulin ($10.7 {\mu}M$), the sandwich protein bound four equivalents of calcium, with half saturation ($K_{0.5}$) observed at a [$Ca^{2+}$] of $8{\mu}M$. However, nicotinamide adenine dinucleotide (NAD) kinase activation property of the sandwich protein was lower than that of calmodulin. The sandwich protein activated NAD kinase, but to only half of the level obtained with calmodulin. The K 0.5 for both calmodulin and the sandwich protein were approximately the same (1-2 nM). Methylation analyses of the sandwich protein show that insertion of calmodulin into DHFR results in a large decrease in methylation. The $V_{max}$ observed with the sandwich protein (95 nmole/min/ml) was only 22% of the value observed with calmodulin (436 nmol/min/ml) in the presence of calcium. Addition of trimethoprim to the reaction significantly inhibited the observed methylation rate. Overall, the data suggest that the insertion of calmodulin into the DHFR structure has little effect on calcium binding by the individual lobes of calmodulin, but may constrain the lobes in a manner that results in altered interaction with the calmodulin-dependent proteins, and severely perturbed the methyltransferase recognition site.

• BMB Reports
• /
• 제31권4호
• /
• pp.405-408
• /
• 1998

Endonuclease-sensitive sites detected by T4 endonuclease V or UvrABC nuclease treatments were compared in the dihydrofolate reductase gene of UV-irradiated Chinese hamster ovary B-11 cells. The number of endonuclease-sensitive sites detected by T4 endonuclease V treatment followed by NaOH denaturation was twice that of formamide denaturation. Repeated treatment of damaged genomic DNA with T4 endonuclease V resulted in no further increase in the number of endonuclease-sensitive sites detected. The numbers of endonuclease-sensitive sites detected by UvrABC nuclease using each denaturation condition were similar. Sequential treatment with the two endonucleases using formamide denaturation resulted in twice the number of endonuclease-sensitive sites detected by treatment of each nuclease alone. Due to a lack of AP endonuclease activity these results suggest the presence of T4 endonuclease V-sensitive sites which could be complemented by alkaline gel separation or by UvrABC nuclease treatment.

• 한국환경성돌연변이발암원학회지
• /
• 제22권1호
• /
• pp.54-59
• /
• 2002

We have used cDNA microarray hybridization to identify gene regulated in response to gamma-irradiation in U-937 cell. The cDNA-chip was composed entirely of 1,000 human cancer related gene including apoptosis and angiogenesis etc. In gamma-irradiated U-937 cell, highly charged protein, ribosomal protein L32, four and a half LIM domains 3, lipocalin 2 (oncogene 24p3) and interleukin 15, ataxia telangiectasia mutated (includes complementation groups A, C and D) genes showed increased level of its transcription, and cell division cycle 25A, dihydrofolate reductase, topoisomerase (DNA) II beta(180kD), kinase suppressor of ras and strarigin genes showed reduced level of its transcription compared to untreated U-937 cell. The significant change of level of transcription was not found in well-known ionizing radiation(IR)-responsive gene, such as transcription factor TP53 and p53 related gene, except ataxia telangiectasia mutated gene.

• 대한수의학회지
• /
• 제36권1호
• /
• pp.57-63
• /
• 1996

Dihydrofolate reductase(DHFR) 억제제는 혈중활성엽산유도체의 농도저하를 초래하는데 이에 대한 기전을 밝히고자 DHFR 억제제의 대표적 약물로서 항암치료에 널리 사용되고 있는 methotrexate를 0.3 및 10mg/kg의 용량으로 랫드에 정맥주사한 후 활성엽산유도체의 담즙배설동태를 검토하였다. 임상적용 용량을 상회하는 용량임에도 불구하고 methotrexate에 의한 환원형엽산유도체의 담즙배설은 유의적 감소를 나타내지 않았다. 이러한 결과는 methotrexate에 의한 엽산유도체의 혈중농도저하가 활성엽산유도체의 담즙배설저하에 직접 관련되지 않는 것을 나타낸다. 따라서 장간순환계가 체내 엽산대사 및 항상성 유지에 중심기구임을 고려해 볼 때 DHFR 억제제에 의한 활성엽산유도체의 혈중농도저하는 담즙배설의 변화에 의한 것 보다는 소화관에 배설된 후 재흡수의 저하에 의해 초래될 가능성이 높은 것으로 사료된다.

• 한국환경성돌연변이발암원학회지
• /
• 제22권4호
• /
• pp.236-242
• /
• 2002

Using agarose-coupled methotrexate, we have successfully isolated two proteins, which have strong interactions with methotrexate. The two proteins were analyzed by Matrix-Assisted Laser Desorption/Ionization Time-of-Flight Mass Spectrometry and identified as carbamoyl-phosphate synthetase 2 and phosphoribosylglycinamide formyltransferase, respectively. Interestingly, both of these two proteins are essential key enzymes in nucleotide biosynthetic pathways, like dihydrofolate reductase, a well-known methotrexate target. We confirmed the specificity of their interactions between methotrexate and two target proteins by the methods of competition binding assay, which were followed by western blotting using antibody against carbamoyl-phosphate synthetase 2 and phosphoribosylglycinamide formyltransferase, respectively. Moreover, we could observe that carbamoyl-phosphate synthetase 2 is overexpressed in methotrexate-resistant MOLT-3 cells comparing with control MOLT-3 cells. This result indicates that carbamoyl-phosphate synthetase 2 may be a novel target of methotrexate in cancer therapy. We propose that chemical proteomics can be a powerful technique to identify target proteins of a chemical.

• Animal cells and systems
• /
• 제1권2호
• /
• pp.323-328
• /
• 1997

We have previously shown that chick muscle extracts contained at least 10 different ubiquitin C-terminal hydrolases (UCHs). In the present studies, one of the enzymes, called UCH-9, was purified by conventional chromatographic procedures using $^{125}l$-labeled ubiquitin-${\alpha}$NH-MHISPPEPESEEEEE HYC (Ub-PESTc) as a substrate. The purified enzyme behaved as a 27-kDa protein under both denaturing and nondenaturing conditions, suggesting that it consists of a single polypeptide chain. It was maximally active at pHs between 7 and 8.5, but showed little or no activity at pH below 6 and above 10. Lice other UCHs, its activity was strongly inhibited by sulfhydryl blocking reagents, such as iodoacetamide, and by Ub-aldehyde. In addition to Ub-PESTc, UCH-9 hydrolyzed Ub-aNH-protein extensions, including Ub-${\alpha}NH$-carboxyl extension protein of 80 amino acids and Ubo-${\alpha}NH$-dihydrofolate reductase. However, this enzyme was not capable of generating free Ub from mono-Ub-${\varepsilon}NH$-protein conjugates and from branched poly-Ub chains that are ligated to proteins through ${\varepsilon}NH$-isopeptide bonds. This enzyme neither could hydrolyze poly-His-tagged di-Ub. These results suggest that UCH-9 may play an important role in production of free Ub and ribosomal proteins from their conjugates.

• 한국생물공학회:학술대회논문집
• /
• 한국생물공학회 2005년도 생물공학의 동향(XVI)
• /
• pp.37-37
• /
• 2005

Human chorionic gonadotropin (hCG) is a heterodimeric glycoprotein hormone consisting of non-covalently linked two subunits, the ${\alpha}$ and ${\beta}$ subunit. It has been used as a infertility drug for ovulation to mimic luteinizing hormone $(LH).^{1)}$ A stable cell line was established by transfection of Rc/CMV-i-dhfr-hCG, expression vector containing hCG ${\alpha}-$ and ${\beta}-genes$, into dihydrofolate reductase-deficient CHO cells and subesquent methotrexate-mediated gene amplification. Anchorage-dependent CHO cells were adapted into a serum-free and/or animal component-free suspension medium through gradual serum weaning for the hCG production. The established cell line showed typical morphological characteristics and growth profile of CHO cells, and could produce FSH with passage-to-passage consistency. The high density perfusion culture of the CHO cells was carried out in Celligen Plus bioreactor equipped with a spin-filter as a internal cell retention device. The cell density reached up to $>1x10^{7}$ cells/ml in less than 7 days and a perfusion-control strategy based on cellular consumption rates of glucose was $established.^{2)}$ Biologically active recombinant hCG was purified by a series of chromatographic steps including anion exchange chromatography and hydrophobic interaction chromatography to homogeneity. The highly purified recombinant hCG was characterized for physicochemical, immunological and biological properties.

• Journal of Microbiology and Biotechnology
• /
• 제23권8호
• /
• pp.1176-1184
• /
• 2013

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.

• Journal of Nutrition and Health
• /
• 제49권2호
• /
• pp.72-79
• /
• 2016

Purpose: The initiation of mandatory folic acid fortification using pteroylmonoglutamic acid (PteGlu) has reduced the rate of congenital malformations. However, it also appears to be responsible for several adverse effects, including increased cancer incidence. This may be related to physicho-chemical characteristics of PteGlu. This study examines the potential effect of high concentrations of PteGlu on a population subjected to mandatory folic acid fortification using an in vitro model. Methods: Caco-2 (colorectal cancer) and MCF7 (breast cancer) cell lines were cultured at 6 different PteGlu concentrations (0, 0.1, 1, 50, 250, and $500{\mu}g/ml$) for 6 days. Cell growth was determined using thiazolyl blue tetrazolium bromide assay. The genotype of dihydrofolate reductase 19bp deletion/insertion (DHFR 19-del) was also scored in cell lines using a restriction fragment length polymorphism technique to examine whether genetic variations may factor in cell proliferation. Results: PteGlu exhibited differential growth promoting properties between cell lines. Caco-2 cells did not show a significant growth difference at low concentrations compared to control, however, at higher concentrations, the growth showed a contrasting trend in the early experimental period, while MCF7 showed enhanced cell growth at all concentrations. The DHFR 19-del genotype differed in the two cell lines. Conclusions: Altered response to PteGlu by Caco-2 and MCF7 may reflect a tissue specific disease aetiology or genotype specific differential enzyme activity, for example by DHFR, to critical levels of PteGlu. As folic acid fortification is a blanket intervention, and DHFR and other enzyme activities vary between individuals, PteGlu intake may have an as yet undefined effect on health. These findings may be relevant when considering mandatory folic acid fortification for disease prevention.

• Biotechnology and Bioprocess Engineering:BBE
• /
• 제6권5호
• /
• pp.332-336
• /
• 2001

When parental Chinese hamster ovary (CHO) cell clones that are capable of producing thrombopoietin (TPO) were subjected to high methotrexate (MTX) concentrations, clonal variations in cell growth were apparent. In the clones that had no significant enhancement in specific TPO productivity (q$\_$Tpo/)when a higher level of MTX was administered, their growth was not depressed significantly nor their cell size changed significantly. On the other hand, those clones that showed a significant-enhancement in q$\_$Tpo/ at higher a MTX dosage, cell growth was depressed initially but recovered during successive sub-cultures. Furthermore, their cell size increased, which suggested that changes in cell size may be indicative of an enhanced q$\_$Tpo/. When the enhancement of the q$\_$Tpo/ of 9 clones after a high MTX dosage was plotted against the extent of the increase of their size, there was a linear correlation (γ$^2$=0.80, p<0.001, ANOVA), which suggested that an enhancement of q$\_$Tpo/ after high MTX administration can be measured by the increase in their cell size. Taken together, our data demonstrate that the selection of amplified CHO cell clones with enhanced q$\_$Tpo/ can be done upon their increased size and growth pattern. This facilitates the development of highly productive recombinant CHO cell lines.