Hyun-Ju Um

Arsenic removal from Vietnamese groundwater using the arsenic-binding DNA aptamer.

Single-stranded DNA aptamers were generated from a random library to remove arsenic from Vietnamese groundwater. On the basis of significant arsenic contamination levels, three areas in Ha Nam province (Vinh Tru, Bo De, and Hoa Hau) and five areas near the Mekong River Delta (MR1-5) were selected as study areas. The aptamers were in vitro selected using an arsenic aptamer affinity column created by immobilizing arsenic on Affi-gel 10 resin. Quantitative analyses of the aptamer candidates Ars-1 to Ars-8 by surface plasmon resonance (SPR) revealed the Ars-3 aptamer to have the highest affinity to arsenate [(As(V)] and arsenite [As(III)] with a dissociation constant (K(d)) of 4.95 +/- 0.31 and 7.05 +/- 0.91 nM, respectively. The specific affinity interactions of the Ars-3 aptamer to arsenic were verified against other heavy metals. After obtaining successful removal results with a laboratory-prepared aqueous arsenic solution, Ars-3 was applied for removal of any arsenic present in the groundwater samples collected from the studied areas in Vietnam. Field results were also successful: various arsenic concentrations ranging from 28.1 to 739.2 microg/L were completely removed after 5 min of incubation with the arsenic-binding aptamer Ars-3.

Analytical bioconjugates, aptamers, enable specific quantitative detection of Listeria monocytogenes

As a major human pathogen in the Listeria genus, Listeria monocytogenes causes the bacterial disease listeriosis, which is a serious infection caused by eating food contaminated with the bacteria. We have developed an aptamer-based sandwich assay (ABSA) platform that demonstrates a promising potential for use in pathogen detection using aptamers as analytical bioconjugates. The whole-bacteria SELEX (WB-SELEX) strategy was adopted to generate aptamers with high affinity and specificity against live L. monocytogenes. Of the 35 aptamer candidates tested, LMCA2 and LMCA26 reacted to L. monocytogenes with high binding, and were consequently chosen as sensing probes. The ABSA platform can significantly enhance the sensitivity by employing a very specific aptamer pair for the sandwich complex. The ABSA platform exhibited a linear response over a wide concentration range of L. monocytogenes from 20 to 2×10(6) CFU per mL and was closely correlated with the following relationship: y=9533.3x+1542.3 (R(2)=0.99). Our proposed ABSA platform also provided excellent specificity for the tests to distinguish L. monocytogenes from other Listeria species and other bacterial genera (3 Listeria spp., 4 Salmonella spp., 2 Vibrio spp., 3 Escherichia coli and 3 Shigella spp.). Improvements in the sensitivity and specificity have not only facilitated the reliable detection of L. monocytogenes at extremely low concentrations, but also allowed for the development of a 96-well plate-based routine assay platform for multivalent diagnostics.

Proteomic analysis of curdlan-producing Agrobacterium sp. in response to pH downshift

During batch cultivation of Agrobacterium sp. ATCC 31750, proteome analysis in response to a pH downshift from 7.0 to 5.5 was carried out using two-dimensional electrophoresis and matrix-assisted laser desorption-ionization-time of flight mass spectrometry. When the pH of the exponentially growing Agrobacterium sp. culture was downshifted to pH 5.5, the synthesis level of 27 intracellular proteins showed significant changes in level over a prolonged period of time compared with the batch culture controlled at pH 7.0. In particular, the intracellular protein level of the beta-1,3-glucan synthase catalytic subunit, UTP-glucose-1-phosphate uridylyltransferase, and phosphoglucomutase, which are key metabolic enzymes in the curdlan biosynthesis pathway, were more than 10-, 3- and 17-times higher in the low pH culture. On the other hand, the level of orotidine5-phosphate decarboxylase (conversion of OMP to UMP) was significantly up-regulated after pH downshift. The accumulation of UMP may direct the metabolic flow towards the biosynthetic route of UTP, which is a key metabolic precursor for UDP-glucose. Therefore, it is possible that increase of cellular metabolic enzymes during pH downshift culture can enhance the metabolic flux of the biosynthesis of key precursor, such as UTP- and UDP-glucose, resulting in an increase in curdlan biosynthesis.

Electrochemically oriented immobilization of antibody on poly-(2-cyano-ethylpyrrole)-coated gold electrode using a cyclic voltammetry.

Using quartz crystal microbalance (QCM) as an immunosensor, this work investigates the contribution of a cyclic voltammetry (CV) on the proper immobilization of antibodies with the aim of enhancing its target recognition and binding ability. Primarily, CV in the range of -0.1 to 0.9 V was applied to form a layer of poly-(2-cyano-ethylpyrrole) (PCEPy) on gold quartz crystal electrode. Then the efficiencies of antibodies (anti-IgG, AIgG) immobilized electrochemically with CV applied in 0-0.65 V were compared to those immobilized via physical adsorption, by observing relative affinity towards AIgG-Fab and AIgG-Fc fragments. The results showed antibody-AIgG-Fab interaction could be enhanced about 4 times when CV is applied (11.2 ± 1.3 vs 41.6 ± 3.4 relative fluorescence unit). On the contrary, physisorbed antibodies showed a higher degree of affinity towards AIgG-Fc indicating inappropriate orientations of physisorbed antibodies. AIgG immobilized PCEPy-gold QC electrode was characterized further for its sensitivity towards a new target bovine albumin with both a QCM and fluorescence measurement. Such electrode exhibited a good sensitivity as well as a large linear dynamic range, from 0.4 μg/ml to 1.0 μg/ml and from 0.5 μg/ml to 10.0 μg/ml, at QCM and fluorescence measurement, respectively.

Immobilization of cross‐linked lipase aggregates onto magnetic beads for enzymatic degradation of polycaprolactone

Candida rugosa lipase was immobilized on amino‐functionalized magnetic supports via cross‐linked enzyme aggregates (CLEA) and used to enhance the enzymatic degradation of polycaprolactone (PCL). The maximum amounts of lipase immobilized on the magnetic beads using glutaraldehyde as a coupling agent were determined to be 33.7 mg/g of beads with an 81% recovery of activity after immobilization. Compared to the free enzyme, the immobilized lipase showed the optimum pH at 1 unit higher (pH 8.0) and also retained its enzymatic activity at higher temperatures. There was 62.9% retention of lipase activity after 30 consecutive reuses, indicating its stability and reusability in aqueous media. Moreover, the immobilized lipase maintained more than 80% of its initial activity during 30 days storage period, while the free lipase lost all under same condition. In addition, the immobilized lipase showed a more than 6‐fold increase in biodegradability over the free lipase when the immobilized lipase was used to degrade PCL in a batch system. Higher thermal and storage stability, as well as good durability after repeated use of the immobilized lipase CLEA, highlights its potential applicability as large scale continuous systems for the enzymatic degradation of PCL. (© 2010 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)

Preventing the formation of positive transcription elongation factor b by human cyclin T1-binding RNA aptamer for anti-HIV transcription

Objective:Development of an innovative antitranscriptional technique for HIV. Design:Systematic evolution of ligands by exponential enrichment (SELEX) technique that can characterize target-specific aptamer was employed to synthesize an aptamer that binds human cyclin T1 (CycT1). When CycT1-binding aptamer interferes the binding of cyclin-dependent kinase 9 (Cdk9) to CycT1, HIV transcription is likely to be discouraged. Methods:Throughout SELEX steps, RNA aptamers having high specific affinity toward CycT1 were characterized. The binding interaction between selected aptamers and CycT1 was analyzed via various techniques. Results:Both qualitative and quantitative analyses revealed Apt4 aptamer, among four candidates, has the highest specific affinity to CycT1. In the presence of Apt4, Cdk9 protein was unable to make interaction with CycT1. Conclusion:A specific RNA aptamer that identifies and binds to CycT1 with high affinity was successfully characterized. As CycT1 plays an important role in HIV transcription, this novel method that interferes and inhibits the transcription of HIV has the potential of being exploited in extended research fields, such as clinical therapy.

The antifouling potentiality of galactosamine characterized from Vibrio vulnificus exopolysaccharide

To gain a better insight into biofilm composition, the exopolysaccharide (EPS) of the Gram-negative bacterium Vibrio vulnificus was studied. Monosaccharide composition analysis of the wild-type and mutant V. vulnificus EPS carried out with Bio-liquid chromatography revealed the presence of d-glucosamine, d-galactose, d-glucose and d-xylose in both strains. d-Galactosamine was found only in the mutant that formed less biofilm compared to its wild-type. The influence of galactosamine on biofilm formation was then studied by adding this substance gradually to six different Gram-negative/positive bacteria associated with various autoinducers. Four bacterial species known to use the autoinducer type-2 signaling system produced less biofilm in the presence of galactosamine. No significant inhibition of biofilm formation was observed in bacteria that produce autoinducer type-1 signal molecules. Galactosamine was also immobilized on polymeric nanofibers to determine its re-usability for the study of biofilm inhibition. The immobilized galactosamine retained >65% of its initial antifouling activity after 10 repeated uses. The results of this study suggest the antifouling role of galactosamine for bacteria that produce AI-2.

The Utilization of Triton X-100 for Enhanced Two-Dimensional Liquid-Phase Proteomics

One of the main challenges in proteomics lies in obtaining a high level of reproducible fractionation of the protein samples. Automated two-dimensional liquid phase fractionation (PF2D) system manufactured by Beckman Coulter provides a process well suited for proteome studies. However, the protein recovery efficiency of such system is low when a protocol recommended by the manufacturer is used for metaproteome profiling of environmental sample. In search of an alternative method that can overcome existing limitations, this study replaced manufacturers buffers with Triton X-100 during the PF2D evaluation of Escherichia coli K12. Three different Triton X-100 concentrations—0.1%, 0.15%, and 0.2%—were used for the first-dimension protein profiling. As the first-dimension result was at its best in the presence of 0.15% Triton X-100, second-dimension protein fractionation was performed using 0.15% Triton X-100 and the standard buffers. When 0.15% Triton X-100 was used, protein recovery increased as much as tenfold. The elution reliability of 0.15% Triton X-100 determined with ribonuclease A, insulin, α-lactalbumin, trypsin inhibitor, and cholecystokinin (CCK) affirmed Triton X-100 at 15% can outperform the standard buffers without having adverse effects on samples. This novel use of 0.15% Triton X-100 for PF2D can lead to greater research possibilities in the field of proteomics.

Toxicoproteomic analysis of bovine aortic endothelial cell under exposure to cigarette smoking extracts

The major health implications of exposure to cigarette smoke that leads to disease development are still being explored. The overall protein expression pattern of bovine aortic endothelial cell (BAEC) has been studied during the treatment with cigarette smoke extract (CSE) by using two-dimensional gel electrophoresis and matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry. Out of an average 950 spots in silver-stained gels, the synthetic levels of 4 proteins were significantly increased, whereas 7 proteins were decreased. The identified proteins have been found to be involved in anti-inflammation (Apolipoprotein A1), energy metabolism (ATP synthase subunit d), signaling pathway (Acetyl-CoA carboxylase; GTP-binding protein), oxidative stress response (Phosphatidylinositol transfer protein beta isoform; phosphatidylinositol transfer protein alpha isoform; Vascular endothelial growth factor), carcinogenesis (Insulin-like growth factor-binding protein 2; Visinin-like protein-1), exocytosis machinery (Synaptosomal-associated protein 25) and immune response (C4b-binding protein). A significantly different protein expression pattern between the control and the CSE-exposed BAEC has been observed and demonstrated on the synthesis level analysis. The systematic approach to the analysis of proteomic responses used as well as the detailed analysis results has been useful in understanding the novel molecular mechanisms for cigarette smoke induced pathological changes. This will help in designing the systemic strategies for the development of new therapeutic approaches to smokerelated diseases.

Comparative analysis of cigarette smoke induced cellular proteome distributions on bovine aortic endothelial cells

Cigarette smoke is a contributory factor for the cardiovascular disease, lung diseases and cancers as the dominant illnesses. The proteomic analysis of bovine aortic endothelial cells (BAECs) exposed to cigarette smoke has been performed in the broad (Non-linear pH 3–11) and narrow (Linear pH 4–7) range by using two-dimensional gel electrophoresis (2-DE). Out of an average 950 spots observed in 2-DE gels under pH 3–11, the expression level of 25 proteins significantly increased with an increase of cigarette smoke extract (CSE) level, whereas 21 proteins were strongly down-regulated. In narrow (4–7) pH range analysis, 80 proteins showed a significant change in expression level as compared with control. Some of the proteins were found to show similar spot intensity patterns in both the linear (4–7) and nonlinear (3–11) pH ranges. Most of the proteins identified in both pH conditions were found to be involved in apoptosis, inflammation, transcription modulator, signal transduction pathway, ROS production, cell proliferation and extracellular structure formation. In addition, extracellular structural proteins also responded to apoptotic signaling as an indicator. The findings of the present study can be used as an early biomarker to indicate the risks of cigarette smoke related diseases and also in the design of new therapeutic and diagnostic approaches to control these diseases.