Jiho Min

Immobilization of lysozyme-CLEA onto electrospun chitosan nanofiber for effective antibacterial applications.

Chitosan (CS) nanofibers with a diameter of 150-200nm were fabricated from a mixed chitosan/poly (vinyl alcohol) (PVA) solution by the electrospinning method. The nascent CS/PVA nanofibers were treated with 0.5M NaOH solution to make stable CS nanofibers by removing PVA under aqueous conditions. Hen egg-white lysozyme was immobilized on electrospun CS nanofibers via cross-linked enzyme aggregates (CLEAs) and used for effective and continuous antibacterial applications. The maximum amount of lysozyme immobilized on the CS nanofibers was determined to be 62.3mg/g of nanofibers under the optimum conditions. The immobilized lysozyme-CLEA retained more than 75.4% of its initial activity after 80 days of storage at room temperature, while the free lysozyme lost all of its activity under the same conditions. In addition, the immobilized lysozyme-CLEA retained more than 76% of its activity after 100 consecutive uses. Finally, the durability of the lysozyme-CLEA immobilized CS nanofibers showed bacteriostasis ratios of 82.4%, 79.8%, 83.4%, and 84.1% after 10 cycles against 4 pathogenic bacteria, viz. Staphylococcus aureus, Bacillus subtilis, Shigella flexneri, and Psedomonas aeruginosa, respectively. These results demonstrated that lysozyme-CLEA immobilized CS nanofibers could be used as a promising material for enhanced and continuous antibacterial applications.

Effects of glyphosate and methidathion on the expression of the Dhb, Vtg, Arnt, CYP4 and CYP314 in Daphnia magna.

In this study, the expression of five stress responsive genes was quantified and analyzed using a semi-quantitative RT-PCR to study the changes in their expression in Daphnia magna after exposure to known pesticides, glyphosate and methidathion. Hemoglobin (Dhb), which was used to show the effect of the oxygen level in the aquatic system, was significantly expressed in D. magna after exposure to glyphosate and methidathion. Additionally, aryl hydrocarbon receptor nuclear translocator (Arnt), a gene related to the metabolism of aryl hydrocarbons, had lower expression levels in D. magna than within the control. CYP4, which was used among cytochrome P450s (CYPs) to show the effects on the fatty acid and steroids metabolisms, was down-regulated in D. magna exposed to glyphosate. However, methidathion affected the expression of CYP314, which was used to show effects of ecdysis, not CYP4 in D. magna. Therefore, glyphosate and methidathion probably caused physiological effects with different patterns in D. magna, especially metabolisms related to CYPs. On the other hand, only vitellogenin (Vtg), which was responsive to the estrogenic potency, did not show any differences in D. magna after exposure to glyphosate or methidathion.

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.

Hypoxia-Induced Endothelial NO Synthase Gene Transcriptional Activation Is Mediated Through the Tax-Responsive Element in Endothelial Cells

Although hypoxia is known to induce upregulation of endothelial NO synthase (eNOS) gene expression, the underlying mechanism is largely unclear. In this study, we show that hypoxia increases eNOS gene expression through the binding of phosphorylated cAMP-responsive element binding (CREB) protein (pCREB) to the eNOS gene promoter. Hypoxia (1% O2) increased both eNOS expression and NO production, peaking at 24 hours, in bovine aortic endothelial cells, and these increases were accompanied by increases in pCREB. Treatment with the protein kinase A inhibitor H-89 or transfection with dominant-negative inhibitor of CREB reversed the hypoxia-induced increases in eNOS expression and NO production, with concomitant inhibition of the phosphorylation of CREB induced by hypoxia, suggesting an involvement of protein kinase A/pCREB-mediated pathway. To map the regulatory elements of the eNOS gene responsible for pCREB binding under hypoxia, we constructed an eNOS gene promoter (−1600 to +22 nucleotides) fused with a luciferase reporter gene [pGL2-eNOS(−1600)]. Hypoxia (for 24-hour incubation) increased the promoter activity by 2.36±0.18-fold in the bovine aortic endothelial cells transfected with pGL2-eNOS(−1600). However, progressive 5′-deletion from −1600 to −873 completely attenuated the hypoxia-induced increase in promoter activity. Electrophoretic mobility shift, anti-pCREB antibody supershift, and site-specific mutation analyses showed that pCREB is bound to the Tax-responsive element (TRE) site, a cAMP-responsive element–like site, located at −924 to −921 of the eNOS promoter. Our data demonstrate that the interaction between pCREB and the Tax-responsive element site within the eNOS promoter may represent a novel mechanism for the mediation of hypoxia-stimulated eNOS gene expression.

Proteomic analysis in Daphnia magna exposed to As(III), As(V) and Cd heavy metals and their binary mixtures for screening potential biomarkers.

In this study, the effects of three widespread heavy metals, As(III), As(V) and Cd, and their binary mixtures on the proteomic profile in D. magna were examined to screen novel protein biomarkers using the two-dimensional gel electrophoresis method (2DE). Ten 20d daphnia were exposed to the LC20 concentrations for each of a total of 8 treatments, including the control, As(III), As(V), Cd, [As(III)+As(V)], [As(III)+Cd], [As(V)+Cd], and [As(III), As(V), Cd], for 24h before protein isolation. Three replicates were performed for each treatment. These protein samples were employed for 2DE experiments with a pH gradient gel strip from pH 3 to pH 10. The protein spots were detected by a silver staining process and their intensities were analyzed by Progenesis software to discover the differentially expressed proteins (DEPs) in response to each heavy metal. A total of 117 differentially expressed proteins (DEPs) were found in daphnia responding to the 8 treatments and mapped onto a 2D proteome map, which provides some information of the molecular weight (MW) and pI value for each protein. All of these DEPs are considered as potential candidates for protein biomarkers in D. magna for detecting heavy metals in the aquatic ecosystem. Comparing the proteomic results among these treatments suggested that exposing D. magna to binary mixtures of heavy metals may result in some complex interactive molecular responses within them, rather than just the simple sum of the proteomic profiles of the individual chemicals, (As(III), As(V), and Cd).

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.

Increased In Vitro Lysosomal Function in Oxidative Stress-Induced Cell Lines

Exposure of mammalian cells to oxidative stress alters lysosomal enzymes. Through cytochemical analysis of lysosomes with LysoTracker, we demonstrated that the number and fluorescent intensity of lysosome-like organelles in HeLa cells increased with exposure to hydrogen peroxide (H2O2), 6-hydroxydopamine (6-OHDA), and UVB irradiation. The lysosomes isolated from HeLa cells exposed to three oxidative stressors showed the enhanced antimicrobial activity against Escherichia coli. Further, when lysosomes that were isolated from HeLa cells exposed by oxidative stress were treated to normal HeLa cells, the viability of the HeLa cells was drastically reduced, suggesting increased in vitro lysosomal function (i.e., antimicrobial activity, apoptotic cell death). In addition, we also found that cathepsin B and D were implicated in increased in vitro lysosomal function when isolated from HeLa cells exposed by oxidative stress. Decrease in cathepsin B activity and increase in cathepsin D activity were observed in lysosomes isolated from HeLa cells after treatment with H2O2, 6-ODHA, or UVB, but cathepsin B and D were not the sole factors to induce cell death by in vitro lysosomal function. Therefore, these studies suggest a new approach to use lysosomes as antimicrobial agents and as new materials for treating cancer cell lines.

Proline reduces the binding of transcriptional regulator ArgR to upstream of argB in Corynebacterium glutamicum

In this study, the ArgR-binding sites on the arg operon Corynbebacterium glutamicum were characterized by in vivo chromatin immunoprecipitation (ChIP). In addition, the ArgR-binding affinity in the presence of glutamate, proline, or arginine was examined to get further information on expression control. The ChIP assay showed that the ArgR protein binds specifically to the upstream regions of argC, argB, argF, and argG. Upon proline supplementation, ArgR-binding affinity was significantly reduced upstream of argB, resulting in increased ornithine production. In contrast, there was no change in the binding affinity of ArgR to the upstream regions of argC, argF, argG, or argB following the addition of glutamate and arginine. These results suggest that the upstream region of argB on the arg operon plays an important role in interacting with ArgR under proline-supplemented conditions and that proline causes an increase in the endogenous level of ornithine by reducing the binding affinity of ArgR to the upstream region of argB.

Enhanced degradation and toxicity reduction of dihexyl phthalate by Fusarium oxysporum f. sp pisi cutinase

Aims:  This research aims to investigate the efficiency of two lipolytic enzymes – fungal cutinase and yeast esterase – upon the biodegradation of dihexyl phthalate (DHP).

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)