• Journal of Microbiology and Biotechnology
• /
• v.19 no.12
• /
• pp.1557-1564
• /
• 2009

Human CDX2 is known as a caudal-related homeodomain transcription factor that is expressed in the intestinal epithelium and is important in differentiation and maintenance of the intestinal epithelial cells. The caudal-related homeobox proteins bind DNA according to a helix-turn-helix structure, thereby increasing the structural stability of DNA. A cancer-tumor suppressor role for Cdx2 has been shown by a decrease in the level of the expression of Cdx2 in colorectal cancer, but the mechanism of transcriptional regulation has not been examined at the molecular level. We developed a large-scale system for expression of the recombinant, novel CDX2, in Escherichia coli. A highly purified and soluble CDX2 protein was obtained in E. coli strain BL21(DE3)RIL and a hexahistidine fusion system using Ni-NTA affinity column, anion exchange, and gel filtration chromatographies. The identity and secondary structure of the purified CDX2 protein were confirmed by MALDI-TOF MS, Western blot, and a circular dichroism analyses. In addition, we studied the DNA-binding activity of recombinant CDX2 by ELISA experiment and isolated human CDX2-binding proteins derived from rat cells by an immobilized GST-fusion method. Three CDX2-binding proteins were found in the gastric tissue, and those proteins were identified to the homeobox protein Hox-D8, LIM homeobox protein 6, and SMC1L1 protein.

• Bulletin of the Korean Chemical Society
• /
• v.32 no.8
• /
• pp.2695-2699
• /
• 2011

${\beta}$ Ketoacyl acyl carrier protein synthase III (KAS III) initiates fatty acid synthesis in bacteria and is a key target enzyme to overcome the antibiotic resistance problem. In our previous study, we found flavonoid inhibitors of Enterococcus faecalis KAS III and proposed three potent antimicrobial flavonoids against Enterococcus faecalis and Vancomycin-resistant Enterococcus faecalis with MIC values in the range of 128-512 ${\mu}g/mL$ as well as high binding affinities on the order from $10^6$ to $10^7\;M^{-1}$. Using these series of flavonoids, we conducted biological assays as well as docking study to find potent flavonoids inhibitors of Staphylococcus aureus KAS III with specificities against Staphylococcus aureus and Methicillin-resistant Staphylococcus aureus. Here, we propose that naringenin (5,7,4'-trihydroxyflavanone) and eriodictyol (5,7,3',4'-tetrahydroxyflavanone) are potent antimicrobial inhibitors of Staphylococcus aureus KAS III with binding affinity of $3.35{\times}10^5$ and $2.01{\times}10^5\;M^{-1}$, respectively. Since Arg38 in efKAS III is replaced with Met36 in saKAS III, this key difference caused one hydrogen bond missing in saKAS III compared with efKAS III, resulting in slight discrepancy in their binding interactions as well as decrease in binding affinities. 4'-OH and 7-OH of these flavonoids participated in hydrogen bonding interactions with backbone carbonyl of Phe298 and Ser152, respectively. In particular, these flavonoids display potent antimicrobial activities against various MRSA strains in the range of 64 to 128 ${\mu}M$ with good binding affinities.

• YAKHAK HOEJI
• /
• v.40 no.6
• /
• pp.734-738
• /
• 1996

The development of the alpha2 isoform of (Na,K)ATPase which is high affinity ouabain receptors was studied in the differentiating nonfusing muscle cell line BC3H-1. T he differentiation process of BC3H-1 cell line was confirmed by 2-dexy-D-[$^3$H] glucose uptake experiment and the quantity of the expression of ${\alpha}_2$ isoform was measured using a whole cell [$^3$H] ouabain-binding assay. Undifferentiated growing BC3H-1 cells, myoblasts, exhibited low levels of insulin-stimulated glucose uptake and [$^3$H] ouabain-binding sites. In contrast, differentiated BC3H-1 cells, myocytes, had a 5.6-fold increase in insulin-stimulated glucose uptake and 5-fold increase in [$^3$H] ouabain-binding sites. Scatchard analysis showed that myocytes developed more [$^3$H] ouabain-binding sites than myoblasts vath a dissociation constant (kd) of 6${\times}10^{-8}$M and capacity of 6.l${\times}10^{-5}$ sites/cell. Therefore. it seems that myoblasts express low levels of ${\alpha}_2$ subunit and probably the majority of ${\alpha}_1$ subunit, whereas myocytes express high levels of ${\alpha}_2$ isoform. The results indicate that the expression of ${\alpha}_2$ isoform is developmentally regulated during differentiation and that BC3H-1 culture system provides an excellent model for the study of differentiation and mechanism of (Na,K)ATPase action in muscle which requires electrical excitability.

• The Journal of Korean Society of Virology
• /
• v.30 no.1
• /
• pp.83-99
• /
• 2000

A defective HIV-1 helper virus DNA, pHyPC, was assembled by deleting the RNA packaging signal, env, nef and the 3'LTR sequences. HIV-1 like virus particles that carry the HIV-1 receptor, CD4 were generated by co expression of pHyPC and plasmid DNAs encoding different chimeric CD4 proteins. The CD4 particles, sharing the CD4 ectodomain, precisely fused to different membrane anchors. CD4(+) particles specifically bound to HIV-1 Env expressing cells, but any signs of infection into these cells were not detected. Binding was only partially blocked by either polyclonal anti-CD4 antibodies or by high concentrations of soluble CD4. Surprisingly, CD4(+) particles also adsorbed to HeLa, CHO, NIH3T3 and COS-7 cells in the absence of HIV-1 Env expression. Adsorption was comparable in strength and speed to the highly specific CD4-Env interaction. CD4(-) particles exhibited only background levels of binding. Cell binding was CD4. dependent, but it was independent of the cell type from which the CD4(+) particles originated. Interestingly, CD4-dependent/Env-independent binding was only found when CD4 was present on virus particles. This suggests that the micro-environment of CD4 on virus particles uniquely expose this new cell binding activity. Its high affinity could explain in part why infection of Env(+) cells by CD4(+) particles was not detected. Further experiments will be required to evaluate whether this strong membrane interaction could represent one step in the multiple-step viral entry process.

• Molecules and Cells
• /
• v.24 no.2
• /
• pp.240-246
• /
• 2007

The ${\beta}2$ integrins on leukocytes play important roles in cell adhesion, migration and phagocytosis. One of the ${\beta}2$ integrins, ${\alpha}X{\beta}2$ (CD11c/CD18), is known to bind ligands such as fibrinogen, Thy-1 and iC3b, but its function is not well characterized. To understand its biological roles, we attempted to identify novel ligands. The functional moiety of ${\alpha}X{\beta}2$, the ${\alpha}X$ I-domain, was found to bind plasminogen, the zymogen of plasmin, with moderate affinity ($1.92{\times}10^{-6}M$) in the presence of $Mg^{2+}$ or $Mn^{2+}$. The ${\beta}D-{\alpha}5$ loop of the ${\alpha}X$ I-domain proved to be responsible for binding, and lysine residues ($Lys^{242}$, $Lys^{243}$) in the loop were the most important for recognizing plasminogen. An excess amount of the lysine analog, 6-aminohexanoic acid, inhibited ${\alpha}X$ I-domain binding to plasminogen, indicating that binding is lysine-dependent. The results of this study indicate that leukocytes regulate plasminogen activation, and consequently plasmin activities, through an interaction with ${\alpha}X{\beta}2$ integrin.

• Natural Product Sciences
• /
• v.25 no.1
• /
• pp.28-33
• /
• 2019

A popular approach for the study of estrogen receptor ${\alpha}$ inhibition is to investigate the protein-protein interaction between the estrogen receptor (ER) and the coactivator surface. In our study, we investigated phytochemicals from Rubus coreanus that were able to disrupt $ER{\alpha}$ and coactivator interaction with an $ER{\alpha}$ antagonist. The E-screen assay and molecular docking analysis were performed to evaluate the effects of the estrogenic activity of R. coreanus extract and its constituents on the MCF-7 human breast cancer cell line. At $100{\mu}g/mL$, R. coreanus extract significantly stimulated cell proliferation ($574.57{\pm}8.56%$). Sanguiin H6, which was isolated from R. coreanus, demonstrated the strongest affinity for the $ER{\alpha}$ coactivator-binding site in molecular docking analysis, with a binding energy of -250.149. The initial results of the study indicated that sanguiin H6 contributed to the estrogenic activity of R. coreanus through the activation of the $ER{\alpha}$ coactivator-binding site.

• The Korean Journal of Nuclear Medicine
• /
• v.29 no.1
• /
• pp.98-104
• /
• 1995

Recently murine monoclonal antibodies have been studied actively for radioimmuno-scintigraphy and radioimmunotherapy, especially on patients with leukemia and lymphoma. In this research, we studied radiolabeling and immunologic characteristics of two in-house anti-leukemic monoclonal antibodies(anti-CALLA & anti-JL-1 antibodies) to make the basis for their clinical application. Each antibody was radiolabeled successfully with $^{99m}Tc$ by pretargeting transchelation method and with $^{125}I$ by lodogen method. We also studied cell binding assay, Scatchard analysis and modulation phenomenon. $^{125}I$ showed 90% labeling efficiency for each anti-body which was satisfactory, but $^{99m}Tc$ showed labeling efficiency below 70%, for which we need better labeling method. In cell binding assay, the immunoreactivity(IR) was low for $^{99m}Tc$-labeled antibodies. Scatchard analysis showed satisfactory data for both binding affinity. The affinity constant and antibody binding sites per cell are around $10^9M^{-1}$ and $10^4$, respectively. There was no modulation phenomenon in cases of $^{125}I$ or $^{99m}Tc$ labeled antibodies. We expect that two anti-leukemic monoclonal antibodies may be useful in diagnosis and therapy for leukemia and lymphoma patients.

• Bulletin of the Korean Chemical Society
• /
• v.34 no.8
• /
• pp.2419-2424
• /
• 2013

Guanosine-5'-diphosphate 3'-diphosphate (ppGpp) serves as alarmone in bacterial stringent responses. In this study, an affinity column was constructed by immobilizing ppGpp to NHS-Sepharose for isolating ppGpp-binding proteins. A novel ppGpp-binding protein, YjgA, was isolated and characterized by MALDI-TOF MS (matrix-assisted laser desorption ionization-time-of-flight mass spectrometry) coupled with two-dimensional gel electrophoresis. YjgA and truncated forms of YjgA were cloned and over-expressed in BL21 (DE3). The binding affinity of YjgA to ppGpp was determined by equilibrium dialysis. The interaction of YjgA with ppGpp was very specific, considering that the dissociation constant of YjgA with ppGpp was measured as $5.2{\pm}2.0{\mu}M$, while the affinities to GTP and GDP were about 60 and 30 times weaker than ppGpp. Expression of yjgA gene in Escherichia coli K-12 MG1655 was examined by reverse transcription polymerase chain reaction (RT-PCR). RT-PCR results revealed that yjgA was expressed from early to late stationary phase. The yjgA deletion mutant exhibited decreased cell number at stationary phase compared to parent strain and the over-expression of YjgA increased the cell number. These results suggested that YjgA might stimulate cell division under stationary phase. In most prokaryotic genome, about half of the protein candidates are hypothetical, that are expected to be expressed but there is no experimental report on their functions. The approach utilized in this study may serve as an effective mean to probe the functions of hypothetical proteins.

• Archives of Pharmacal Research
• /
• v.18 no.2
• /
• pp.90-99
• /
• 1995

The effect of protein kinase C inhibitors, sturosporine and 1-(5-isoquinolinyl sulfonyl)-2-methyl piperazine(H7) on in vitro differentiation of erythroid progenitor cells which were isolated from spleens of mice infected with the anemia-inducing strain of Friend virus were examined. Erythropoietin-mediated differentitation of erythroid progenitor cells, as determined by the incorporation of $^{59}Fe$ into protoporphyrin, was inhibited by staurosporine and H7 in a concentration -dependent manner. Scatchard analysis of the $^3H-phorbol-12$, 13-dibutyrate binding to erythroid progenitor cells revealed that at the high affinity sites the dissociation constant was 22nM and the maximum number of $^3H-phorbol-12$, 13-dibutyrate binding to erythroid progenitor cells revealed that at the high affinity sites the dissociation constant was 22nM and the maximum number of $^3H-phorbol-12$, 13-dibutyrate binding sites per cell was approximately $3.7\times10^5$. Cytosonic protein kinase C was isolated from erthroid progenitor cells and then purified by sequential column chromatogrphy. Two isoforms of protein kinase C were found. Photoaffinity labeling of the purified protein kinase C samples with $^3H-phorbol-12$12-myristate 13-acetate followed by analysis of SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and autofluorography showed radiolabeled 82-KDa pepticles. Rediolabeling of the 82-KDa peptides with $^3H-phorbol-12$myristate 13-acete was almost completely blocked by excess unlabeled phorbol 12-myristate 13-acetate was almost 12-muristate 13-acetate-promoted phosphorylation with the puyrified protein kinase C samples showed that the phosphorylation of 82-KDa peptides was increased as the concentration of phorbol 12-myristate 13-acetate was increased from $10^{-8}M{\;}to{\;}10^{-4}$M. In light of the findings that erythroid progenitor cells possessed an abundance of protein kinase C and that stauroporine and H7 inhibited erythroid differentiation, it seemed likely that protein kinase C would play a role in the erythroid progenitor cell development.

• Clinical and Experimental Reproductive Medicine
• /
• v.23 no.3
• /
• pp.247-268
• /
• 1996

It has been known for a long time that gonadotropins and steroid hormones play a pivotal role in a series of reproductive biological phenomena including the maturation of ovarian follicles and oocytes, ovulation and implantation, maintenance of pregnancy and fetal growth & development, parturition and mammary development and lactation. Recent investigations, however, have elucidated that in addition to these classic hormones, multiple growth factors also are involved in these phenomena. Most growth factors in reproductive organs mediate the actions of gonadotropins and steroid hormones or synergize with them in an autocrine/paracrine manner. The insulin-like growth factor(IGF) system, which is one of the most actively investigated areas lately in the reproductive organs, has been found to have important roles in a wide gamut of reproductive phenomena. In the present communication, published literature pertaining to the intrauterine IGF system will be reviewed preceded by general information of the IGF system. The IGF family comprises of IGF-I & IGF-II ligands, two types of IGF receptors and six classes of IGF-binding proteins(IGFBPs) that are known to date. IGF-I and IGF-II peptides, which are structurally homologous to proinsulin, possess the insulin-like activity including the stimulatory effect of glucose and amino acid transport. Besides, IGFs as mitogens stimulate cell division, and also play a role in cellular differentiation and functions in a variety of cell lines. IGFs are expressed mainly in the liver and messenchymal cells, and act on almost all types of tissues in an autocrine/paracrine as well as endocrine mode. There are two types of IGF receptors. Type I IGF receptors, which are tyrosine kinase receptors having high-affinity for IGF-I and IGF-II, mediate almost all the IGF actions that are described above. Type II IGF receptors or IGF-II/mannose-6-phosphate receptors have two distinct binding sites; the IGF-II binding site exhibits a high affinity only for IGF-II. The principal role of the type II IGF receptor is to destroy IGF-II by targeting the ligand to the lysosome. IGFs in biological fluids are mostly bound to IGFBP. IGFBPs, in general, are IGF storage/carrier proteins or modulators of IGF actions; however, as for distinct roles for individual IGFBPs, only limited information is available. IGFBPs inhibit IGF actions under most in vitro situations, seemingly because affinities of IGFBPs for IGFs are greater than those of IGF receptors. How IGF is released from IGFBP to reach IGF receptors is not known; however, various IGFBP protease activities that are present in blood and interstitial fluids are believed to play an important role in the process of IGF release from the IGFBP. According to latest reports, there is evidence that under certain in vitro circumstances, IGFBP-1, -3, -5 have their own biological activities independent of the IGF. This may add another dimension of complexity of the already complicated IGF system. Messenger ribonucleic acids and proteins of the IGF family members are expressed in the uterine tissue and conceptus of the primates, rodents and farm animals to play important roles in growth and development of the uterus and fetus. Expression of the uterine IGF system is regulated by gonadal hormones and local regulatory substances with temporal and spatial specificities. Locally expressed IGFs and IGFBPs act on the uterine tissue in an autocrine/paracrine manner, or are secreted into the uterine lumen to participate in conceptus growth and development. Conceptus also expresses the IGF system beginning from the peri-implantation period. When an IGF family member is expressed in the conceptus, however, is determined by the presence or absence of maternally inherited mRNAs, genetic programming of the conceptus itself and an interaction with the maternal tissue. The site of IGF action also follows temporal (physiological status) and spatial specificities. These facts that expression of the IGF system is temporally and spatially regulated support indirectly a hypothesis that IGFs play a role in conceptus growth and development. Uterine and conceptus-derived IGFs stimulate cell division and differentiation, glucose and amino acid transport, general protein synthesis and the biosynthesis of mammotropic hormones including placental lactogen and prolactin, and also play a role in steroidogenesis. The suggested role for IGFs in conceptus growth and development has been proven by the result of IGF-I, IGF-II or IGF receptor gene disruption(targeting) of murine embryos by the homologous recombination technique. Mice carrying a null mutation for IGF-I and/or IGF-II or type I IGF receptor undergo delayed prenatal and postnatal growth and development with 30-60% normal weights at birth. Moreover, mice lacking the type I IGF receptor or IGF-I plus IGF-II die soon after birth. Intrauterine IGFBPs generally are believed to sequester IGF ligands within the uterus or to play a role of negative regulators of IGF actions by inhibiting IGF binding to cognate receptors. However, when it is taken into account that IGFBP-1 is expressed and secreted in primate uteri in amounts assessedly far exceeding those of local IGFs and that IGFBP-1 is one of the major secretory proteins of the primate decidua, the possibility that this IGFBP may have its own biological activity independent of IGF cannot be excluded. Evidently, elucidating the exact role of each IGFBP is an essential step into understanding the whole IGF system. As such, further research in this area is awaited with a lot of anticipation and attention.