• Parasites, Hosts and Diseases
• /
• v.46 no.4
• /
• pp.209-216
• /
• 2008

A monoclonal antibody against Toxoplasma gondii of Tg556 clone (Tg556) blotted a 29 kDa protein, which was localized in the dense granules of tachyzoites and secreted into the parasitophorous vacuolar membrane (PVM) after infection to host cells. A cDNA fragment encoding the protein was obtained by screening a T. gondii cDNA expression library with Tg556, and the full-length was completed by 5'-RACE of 2,086 bp containing an open reading frame (ORF) of 669 bp. The ORF encoded a polypeptide of 222 amino acids homologous to the revised GRA3 but not to the first reported one. The polypeptide has 3 hydrophobic moieties of an N-terminal stop transfer sequence and 2 transmembrane domains (TMD) in posterior half of the sequence, a cytoplasmic localization motif after the second TMD and an endoplasmic reticulum (ER) retrival motif in the C-terminal end, which suggests GRA3 as a type III transmembrane protein. With the ORF of GRA3, yeast two-hybrid assay was performed in HeLa cDNA expression library, which resulted in the interaction of GRA3 with calcium modulating ligand (CAMLG), a type II transmembrane protein of ER. The specific binding of GRA3 and CAMLG was confirmed by glutathione S-transferase (GST) pull-down and immunoprecipitation assays. The localities of fluorescence transfectionally expressed from GRA3 and CAMLG plasmids were overlapped completely in HeLa cell cytoplasm. In immunofluorescence assay, GRA3 and CAMLG were shown to be co-localized in the PVM of host cells. Structural binding of PVM-inserted GRA3 to CAMLG of ER suggested the receptor-ligand of ER recruitment to PVM during the parasitism of T. gondii.

• Proceedings of the Korean Institute of Electrical and Electronic Material Engineers Conference
• /
• 2001.07a
• /
• pp.757-760
• /
• 2001

We have used the random fluidic self-assembly (RFSA) technique based on the chip pattern of hydrophobic self-assembly layers to assemble microfabricated particles onto the chip pattern. Immobilization of DNA, fabrication of the particles and the chip pattern, arrangement of the particles on the chip pattern, and recognition of each using DNA fluorescence measurement were carried out. Establishing the walls, the arrangement stability of the particles was improved. Each DNA is able to distinguish by using the lithography process on the particles. Advantages of this method are process simplicity, wide applicability and stability. It is thought that this method can be applicable as a new fabrication technology to develop a minute integration type biosensor microarray.

• Biotechnology and Bioprocess Engineering:BBE
• /
• v.8 no.4
• /
• pp.263-267
• /
• 2003

Magnetic nanoparticles prepared from an alkaline solution of divalent and trivalent iron ions could covalently bind protein via the activation of Nethyl-N-(3-dimethylaminopropyl) carbodiimide (EDC). Trypsin and avidin were taken as the model proteins for the formation of protein-nanoparticle conjugates. The immobilized yield of protein increased with molar ratio of EDC/nanoparticie. Higher concentrations of added protein could yield higher immobilized protein densities on the particles. In contrast to EDC, the yields of protein immobilization via the a ctivation of cyanamide were relatively lower. Nanoparticles bound with avidin could attach a single-stranded DNA through the avidin-biotin interaction and hybridize with a DNA probe. The DNA hybridization was confirmed by fluorescence microscopy observations. Immobilized DNA on nanoparticles by this technique may have widespread applicability to the detection of specific nucleic acid sequence and targeting of DNA to particular cells.

• BMB Reports
• /
• v.43 no.6
• /
• pp.407-412
• /
• 2010

Homeodomain (HD) is a highly conserved DNA-binding domain composed of helix-turn-helix motif. Drosophila Vnd (Ventral nervous system defective) containing HD acts as a regulator to either enhance or suppress gene expression upon binding to its target sequence. In this study, kinetic analysis of Vnd binding to DNA was performed. The result demonstrates that DNA-binding affinity of the recombinant protein containing HD and NK2-specific domain (NK2-SD) was higher than that of the full-length Vnd. To access whether phosphorylation sites within HD and NK2-SD affect the interaction of the protein with the target sequence, alanine substitutions were introduced. The result shows that S631A mutation within NK2-SD does not contribute significantly to the DNA-binding affinity. However, S571A and T600A mutations within HD showed lower affinity for DNA binding. In addition, DNA-binding analysis using embryonic nuclear protein also demonstrates that Vnd interacts with other nuclear proteins, suggesting the existence of Vnd as a complex.

• Proceedings of the KIEE Conference
• /
• 2006.07c
• /
• pp.1315-1316
• /
• 2006

Microarray-based DNA chips provide an architecture for multi-analyte sensing. In this paper, we report a new approach for DNA chip microarray fabrication. Multifunctional DNA chip microarray was made by immobilizing many kinds of biomaterials on transducers (particles). DNA chip microarray was prepared by randomly distributing a mixture of the particles on a chip pattern containing thousands of m-scale sites. The particles occupied a different sites from site to site. The particles were arranged on the chip pattern by the random fluidic self-assembly (RFSA) method, using a hydrophobic interaction for assembly.

• Proceedings of the KIEE Conference
• /
• 2004.11a
• /
• pp.271-274
• /
• 2004

Microarray-based DNA chips provide an architecture for multi-analyte sensing. In this paper, we report a new approach for DNA chip microarray fabrication. Multifunctional DNA chip microarray was made by immobilizing many kinds of biomaterials on transducers (particles). DNA chip microarray was prepared by randomly distributing a mixture of the particles on a chip pattern containing thousands of m-scale sites. The particles occupied a different sites from site to site. The particles were arranged on the chip pattern by the random fluidic self-assembly (RFSA) method, using a hydrophobic interaction for assembly.

• Journal of Radiation Industry
• /
• v.5 no.3
• /
• pp.243-247
• /
• 2011

In this study, a convenient method for the selective immobilization of single strand DNA (ssDNA) on a polymer surface was described. A positively charged polyelectrolyte, poly(diallyldimethylammonium chloride) (PDDA), was spin-coated on a tissue culture petridish and the micropatterns of the PDDA were formed by selective ion implantation through a pattern mask. The surface property of the implanted PDDA was investigated by using a surface profiler and FT-IR spectrometer. Cy3-labeled ssDNA was selectively immobilized on the PDDA patterns through ionic interaction and thus, well-defined ssDNA patterns were obtained.

• Bulletin of the Korean Chemical Society
• /
• v.19 no.4
• /
• pp.449-457
• /
• 1998

We compared various spectroscopic properties of a norfloxacin-single stranded DNA complex with those of norfloxacin-double stranded DNA complex. Norfloxacin binds to both double-and single stranded DNA, and we observed the following spectroscopic changes for both complexes: hypochromism in the norfloxacin absorption region in the absorption spectrum, the characteristic induced CD spectrum consisting of a weak positive band at 323 nm and a strong positive band at 280-300 nm followed by a negative band in the 260 nm region, a strong decrease in the fluorescence intensity and a red-shift in the fluorescence emission spectrum, and shorter fluorescence decay times. These results indicate that the environments of the bound norfloxacin in both DNAs are similar, although the equilibrium constant of the norfloxacin-single stranded DNA was twice as high as the norfloxacin-double stranded DNA complex. Both complexes were thermodynamically favored with similar negative Δ$G^o$. Negative Δ$H^o$ terms contribute to these spontaneous reactions; Δ$S^o$ term was unfavorable.

• Journal of Applied Biological Chemistry
• /
• v.57 no.3
• /
• pp.259-265
• /
• 2014

Mitochondrial DNA (mtDNA)-depleted (${\rho}^0$) cells are often used as mtDNA recipients to study the interaction between the nucleus and mitochondria in mammalian cells. Therefore, it is crucial to obtain mtDNA-depleted cells with many different nuclear backgrounds for the study. Here, we demonstrate a rapid and reliable method to isolate mammalian mtDNA-depleted cells involving treatment with the antimitochondrial agents ethidium bromide (EtBr) and 2',3'-dideoxycytidine (ddC). After a short exposure to EtBr or ddC, followed by rapid clonal isolation, we were able to generate viable mtDNA-depleted cells from mouse and human cells and were able to successfully repopulate them with exogenous mitochondria from platelets isolated from mouse and human blood samples. These mtDNA-depleted cells can be used to characterize the nuclear mitochondrial interactions and to study mtDNA-associated defects in mammalian cells. Our method of isolating mtDNA-depleted cells is practical and applicable to a variety of cell types.

• Bulletin of the Korean Chemical Society
• /
• v.30 no.5
• /
• pp.1031-1034
• /
• 2009

Fluorescence of quinolones including norfloxacin, ciprofloxacin and S- and R-ofloxacin is quenched upon association with single and double-stranded DNA (ss- and ds-DNA). The ratios of fluorescence intensity in the presence of DNA to its absent were plotted with respect to the DNA concentration to construct the Stern-Volmer plot. The slope of the Stern-Volmer plot become larger as the temperature is lowered, ensuring that the fluorescence quenching is static process, i.e., the fluorescence is quenched by formation of the non-fluorescent complex between quinolone and DNA. In the static quenching mechanism, the quenching constant which is equivalent to the slope of the Stern-Volmer plot, is considered as the equilibrium constant for the association of quinolones and DNA. From the temperature-dependent equilibrium constant, ${\Delta}H^0\;and\;{\Delta}S^0$ was obtained using the van’t Hoff relation. In general, association of the quinolone with ds- as well as ss-DNA is energetically favorable (an exothermic) process while the entropy change was unfavorable. Due to the steric effect of the substituents, the effect of the quinolone ring is smaller on the ss-DNA compared to ds-DNA.