Biomedical subjects

Man Bock Gu

Publications and source records attributed to Man Bock Gu.

At least 19 recordsLinked to original sources

PubMed2005

Biodegradation of dipropyl phthalate and toxicity of its degradation products: a comparison of Fusarium oxysporum f. sp. pisi cutinase and Candida cylindracea esterase.

The efficiency of two lypolytic enzymes (fungal cutinase, yeast esterase) in the degradation of dipropyl phthalate (DPrP) was investigated. The DPrP-degradation rate of fungal cutinase was surprisingly high, i.e., almost 70% of the initial DPrP (500 mg/l) was decomposed within 2.5 h and nearly 50% of the degraded DPrP disappeared within the initial 15 min. With the yeast esterase, despite the same concentration, more than 90% of the DPrP remained even after 3 days of treatment. During the enzymatic degradation of DPrP, several DPrP-derived compounds were detected and time-course changes in composition were also monitored. The final chemical composition after 3 days was significantly dependent on the enzyme used. During degradation with fungal cutinase, most DPrP was converted into 1,3-isobenzofurandione (IBF) by diester hydrolysis. However, in the degradation by yeast esterase, propyl methyl phthalate (PrMP) was produced in abundance in addition to IBF. The toxic effects of the final degradation products were investigated using various recombinant bioluminescent bacteria. As a result, the degradation products (including PrMP) from yeast esterase severely caused oxidative stress and damage to protein synthesis in bacterial cells, while in the fungal cutinase processes, DPrP was significantly degraded to non-toxic IBF after the extended period (3 days).

Biodegradation, Environmental↗

PubMed2005

Multiple and simultaneous detection of specific bacteria in enriched bacterial communities using a DNA microarray chip with randomly generated genomic DNA probes.

A DNA microarray chip for detecting the presence of specific bacterial strains was developed using random genomic probes derived from genomic DNA, i.e., without any sequence information. Thirteen bacteria from different genuses were selected as targets. For the fabrication of the random genomic probes, genomic DNA from pure cultures of each bacterium was fractionated using several pairs of restriction endonucleases. After size fractionation of the genomic DNA fragments, random genomic libraries for each bacterium were constructed. From the library, specific probes were amplified by PCR and the probes were affixed to a slide glass to fabricate the DNA microarray chip. The results from tests with pure and mixed cultures of the bacteria used in the fabrication of the chips showed specific responses and only a small portion of cross-hybridization. This DNA microarray chip was also tested to detect the presence of specific bacteria in mixed populations. In these tests, it was demonstrated that this system provided a fast and specific response to the presence of bacterial species in mixed samples, even in activated sludge samples. This indicates that any DNA microarray chip for the detection of specific bacteria can be fabricated using the same protocols as presented in this study without requiring any genus level sequence information from pure isolates.

Bacterial Typing Techniques↗

PubMed2005

Immobilization as a technical possibility for long-term storage of bacterial biosensors.

For applications in field experiments, the recombinant strain Salmonella typhimurium TA1535 was immobilized to permit its immediate utilization after long storage periods. Salmonella typhimurium TA1535 cells contain the plasmid that has an inducible SOS promoter fused to a promoterless luxCDABFE operon from Photobacterium leiognathi. The induction of bioluminescence occurs in the presence of the DNA-damaging agent mitomycin C which stimulates the bacterial SOS response. Early stationary phase cells were immobilized at a cell concentration of 10(10) CFU/ml in microtiter plates and stored up to 6 weeks at 4 degrees C in a sealed container. Even after 4 weeks of storage, the bioluminescence kinetics and yield in response to different concentrations of mitomycin C were not significantly different from those of freshly prepared samples.

Biosensing Techniques↗

PubMed2005

An integrated mini biosensor system for continuous water toxicity monitoring.

An integrated water toxicity monitoring system that uses recombinant bioluminescent bacteria was successfully developed for the continuous monitoring and classification of toxicities present in water. This system consists of four channels arranged horizontally inside of a cylinder, with each channel having two small bioreactors that are vertically connected to each other to maintain a separation of the culture reactor from test reactor. This system is easily handled and installed, making its application in the field a potential reality. As well, it performed stably and continuously due to the vertical separation of the culture reactor from the test reactor and a long term operation was also performed because of its small working volume, i.e., only 1 ml for the 1st bioreactor and 2 ml for the 2nd. During an operation with four strains, i.e., EBHJ2, DP1, DK1 and DPD2794, which are responsive to superoxide damage (EBHJ2 and DP1), hydrogen peroxide (DK1), and DNA damage (DPD2794), the O.D. and bioluminescence of the bacterial cultures inside the system were constant when no chemical was injected. However, with the addition of paraquat, hydrogen peroxide or mitomycin C, the bioluminescent responses of the strains were found to be dose-dependent to different concentrations of these chemicals.

Biological Assay↗

PubMed2005

A cell array biosensor for environmental toxicity analysis.

In this study, a cell-based array technology that uses recombinant bioluminescent bacteria to detect and classify environmental toxicity has been implemented to develop two biosensor arrays, i.e., a chip and a plate array. Twenty recombinant bioluminescent bacteria, having different promoters fused with the bacterial lux genes, were immobilized within LB-agar. About 2 microl of the cell-agar mixture was deposited into the wells of either a cell chip or a 384-well plate. The bioluminescence (BL) from the cell arrays was measured with the use of highly sensitive cooled CCD camera that measured the bioluminescent signal from the immobilized cells and then quantified the pixel density using image analysis software. The responses from the cell arrays were characterized using three chemicals that cause either superoxide damage (paraquat), DNA damage (mitomycin C) or protein/membrane damage (salicylic acid). The responses were found to be dependent upon the promoter fused upstream of the lux operon within each strain. Therefore, a sample's toxicity can be analyzed and classified through the changes in the BL expression from each well. Moreover, a time of only 2 h was needed for analysis, making either of these arrays a fast, portable and economical high-throughput biosensor system for detecting environmental toxicities.

Arabidopsis Proteins↗

PubMed2005

A multi-channel continuous water toxicity monitoring system: its evaluation and application to water discharged from a power plant.

A multi-channel continuous water toxicity monitoring system was, after confirming the systems' performance, implemented to samples of water discharged from power plants to detect and classify their toxicity using several recombinant bioluminescent bacteria. Each channel of the system is composed of a series of two mini-bioreactors to enable a continuous operation, i.e., without system interruption due to highly toxic samples. A different recombinant bacterial strain was present in each channel: DPD2540 (fabA::lux CDABE), DPD2794 (recA::luxCDABE), and TV1061 (grpE::luxCDABE), which are induced by cell membrane-, DNA-, and protein-damaging agents, respectively. GC2 (lac::luxCDABE) is a constitutive strain, whose bioluminescence is reduced by an increase in cellular toxicity. Phenol and mitomycin C (MMC) were used for evaluating the system's performance to detect toxic chemicals. These samples were injected into the second mini-bioreactor according to a step or bell-curve manner. The field samples used in this study were obtained from the water discharged from two different power plants in Korea--from a nuclear power plant and a thermo-electronic power plant--and were injected into the second mini-bioreactor to initiate the toxicity test. Each channel showed specific bioluminescent (BL) response profiles due to the toxic compounds present in the water samples. Comparing the BL signals between the standard toxic chemical samples and discharged water samples, the equivalent toxicity of the field water could be estimated. Finally, it was proved that this novel continuous toxicity monitoring system can be used as an alternative tool for the quick monitoring and control of water quality, as well as aid in the setting up of a new monitoring strategy to protect the source of tap water and in the prevention of polluted water discharge.

Alkylating Agents↗

PubMed2005

Construction and evaluation of nagR-nagAa::lux fusion strains in biosensing for salicylic acid derivatives.

The NagR protein is a response regulatory protein found in the bacterium Ralstonia sp. U2 that is involved in sensing for salicylic acid and the subsequent induction of the operon just upstream of its gene. The genes encoded for in this operon are involved in the degradation of salicylic acid. Escherichia coli strain RFM443 carrying a fusion of the Photorhabdus luminescens luxCDABE operon with the nagR gene and upstream region of the nagAa gene was constructed and characterized with respect to its optimum temperature, its response time and kinetics, and its ability to detect numerous benzoic acid derivatives. Although capable of detecting 0.5 mM salicylic acid at any temperature between 28 and 40 degrees C, this E. coli strain, labeled DNT5, showed its greatest relative activity at 30 degrees C, i.e., the temperature at which the largest induction was seen. Furthermore, experiments done with numerous benzoic acid derivatives found the NagR protein to be responsive to only a few of the compounds tested, including salicylic acid and 3-methyl salicylic acid, and acetyl salicylic acid was the strongest inducer. The lower limits of detection for these compounds with E. coli strain DNT5 were also established, with the native inducer, salicylic acid, giving the most sensitive response and detectable down to a concentration of about 2 microM. A second lux fusion plasmid was also constructed and transformed into an NahR background, Pseudomonas putida KCTC1768. Within this strain, NAGK-1768, the supplemental activity of the NahR protein on the nagAa promoter, was shown to extend both the range of chemicals detected and the sensitivity.

Benzene↗

PubMed2004

Development of a DNA microarray chip for the identification of sludge bacteria using an unsequenced random genomic DNA hybridization method.

A tool, based upon the DNA microarray chip, for the identification of specific bacteria from activated sludge, using the hybridization of genomic DNA with random probes, is described. This chip was developed using the genomic DNAs from Gordonia amarae, the natural filamentous actinomycete that causes sludge foaming and bulking, as well as a nonfilamentous floc forming bacterium (Zoogloea ramigera) and the skin pathogen Mycobacterium peregrinum without any sequence information. The sets of target probes on amine-coated glass were made from a genomic library, constructed with PCR products derived from randomly fragmented genomic DNAs extracted from pure cultures of the three strains. Initial hybridization results, when pure cultures were employed, showed the specificity of the probes as well as the resolution of the system, demonstrating the capabilities of this system to identify specific bacterial strains. The microarray was also tested for its ability to distinguish specific bacteria from among mixed bacterial communities, such as in sludge, soil, or spiked genomic DNA samples. The results showed that the probes are specific, with only mild cross-hybridization occurring in a small number of cases. Furthermore, the chip clearly discriminated the presence of all three strains when they were present alone or together within mixed samples. Moreover, using the spot intensity and DNA hybridization kinetics, the starting genomic DNA concentrations could be estimated relatively well, which would make it possible to predict the number of specific bacteria present within the test samples. Therefore, the random genomic hybridization approach, i.e., without any sequence information available for the probes, is a practical protocol for the identification of and screening for specific bacteria within any complex bacterial community from the environmental samples, such as in activated sludge, although the possibility of cross-hybridization may still exist.

Bacteria↗

PubMed2004

Enhancement of the multi-channel continuous monitoring system through the use of Xenorhabdus luminescens lux fusions.

The enhancement of the multi-channel continuous toxicity monitoring system developed previously was studied. To achieve better and more stable results from the system, the use of thermo-lux fusion strains that express the luxCDABE genes from Xenorhabdus luminescens was evaluated. A total of six recombinant Escherichia coli strains with the promoters from three oxidative-stress responsive genes, i.e. the katG, sodA and pqi-5 genes, fused to either the lux genes from Vibrio fischeri or X. luminescens were characterized and their responses to different chemicals compared. It was found that the basal level bioluminescence (BL) from the thermo-lux fusion strains was always higher while that of the V. fischeri lux strains were always near or below the lower limit of detection of the system. For example, the katG::V. fischeri lux strain, DPD2511, gave no discernible response due to its low level expression while a fusion of the katG promoter with the X. luminescens lux operon was clearly responsive and capable of detecting hydrogen peroxide down to about 1 ppm. The use of the thermo-lux strains found them to be as sensitive as the V. fischeri lux strains while providing a brighter, more stable basal level bioluminescence, making the analysis and monitoring of water-borne toxicity more reliable.

Aliivibrio fischeri↗

PubMed2004

Sphingomonas sp. strain SB5 degrades carbofuran to a new metabolite by hydrolysis at the furanyl ring.

Microorganisms capable of degrading carbofuran were isolated from soils and examined for the degradation of this pesticide at ring structure. An isolate that could degrade carbofuran and carbofuran-7-phenol was selected for further studies. The 16S rRNA analysis results showed that the isolate belongs to the genus of Sphingomonas, close to dioxin and dicamba degraders, and is named Sphingomonas sp. SB5. SB5 did not show any similarity of 16S rRNA to known carbofuran degraders. When time-course degradation of carbofuran by SB5 was examined by solvent extraction combined with liquid chromatographic analysis, almost complete disappearance of carbofuran was observed within 12 h, giving several accumulative metabolites. Bacterial cultures incubated with carbofuran-7-phenol suggested that the accumulated metabolites were derived from carbofuran-7-phenol. The control without SB5 and kanamycin-treated SB5 did not show any metabolite, suggesting a biological involvement in the degradation of carbofuran. GC/MS and LC/MS analyses identified 2-hydroxy-3-(3-methylpropan-2-ol) phenol as one of the accumulated metabolites, suggesting that the strain SB5 could degrade carbofuran-7-phenol by hydrolysis at the furanyl ring. This is the first report to identify 2-hydroxy-3-(3-methylpropan-2-ol) phenol as a new product derived biologically from carbofuran-7-phenol.

Carbofuran↗

PubMed2004

Construction and characterization of novel dual stress-responsive bacterial biosensors.

Using the genes for the green fluorescence protein and Xenorhabdus luminescens luciferase operon and the promoters for the recA and katG genes, two stress-responsive Escherichia coli biosensor strains have been constructed that can individually or concurrently respond to oxidative and genotoxic conditions. Strain DUO-1 carries the pRGDK1 plasmid, which has the recA::GFPuv4 and katG::luxCDABE fusion genes oriented divergently with each other, while in DUO-2, i.e., pRGDK2, they are in a tandem orientation, with the recA promoter showing run-though transcription of the katG::luxCDABE fusion. These two strains and their responses were characterized using several known hydroxyl radical-forming chemicals, e.g., hydrogen peroxide and cadmium chloride, along with some genotoxins, e.g., mitomycin C and methyl-N-nitro-N-nitrosoguanidine, and some general toxicants. Both strains showed an induction of green fluorescent protein (GFP) and bioluminescence when they experienced DNA and oxidative damage, respectively, while the tandem orientation of the two fusion genes within DUO-2 allowed it to also sensitively respond to genotoxins via the production of bioluminescence. However, the characteristics of DUO-2's bioluminescent response to each stress were easily distinguishable, making it useful for the detection of both stresses. Furthermore, tests with mixtures of chemicals showed that both DUO-1 and DUO-2 were responsive when chemicals causing oxidative or genotoxic stress were present as a single chemical or within complex chemical mixtures.

Antibiotics, Antineoplastic↗

PubMed2004

Whole-cell-based biosensors for environmental biomonitoring and application.

A variety of whole-cell-based biosensors has been developed using numerous native and recombinant biosensing cells. The use of reporter genes, for example bacterial luciferase and gfp, to monitor gene expression is discussed in terms of each reporters' benefits and disadvantages, including their possible use on-line, their sensitivity, the need for extra substrate, etc. All biosensing cells in use can be classified into two groups in terms of their biosensing mechanisms--constitutive expression and stress- or chemical-specific inducible expression. In this review several examples of each are presented and discussed. The use of recombinant whole-cell biosensors in the field requires three components--biosensing cells, a measurement device, and a signal-transducing apparatus, the last two depending on the first and the final applications of the system. The use of different immobilization techniques in several studies to maintain the cells and their viability is also discussed, in particular their use in the development of both high-throughput and chip-based biosensing systems. Finally the application of whole-cell-based biosensors to different environmental media, such as water, soil, and atmospheric monitoring is discussed; particular attention is given to their use for detection of various stressors, including dioxins, endocrine-disrupting chemicals, and ionizing radiation.

Biosensing Techniques↗

PubMed2003

Gamma-radiation dose-rate effects on DNA damage and toxicity in bacterial cells.

In order to investigate the relationship between radiation dose-rate and bacterial DNA damage as well as general cellular toxicity, two recombinant Escherichia coli strains, DPD2794 and GC2 were used. Following gamma-ray irradiation, these bioluminescent bacteria showed quantitative stress responses in terms of DNA damage and general toxicity depending on the dose rates of energy deposition, i.e. dose-rate of radiation. In addition, an inverse relationship was found, at lower dose rates between 0.5 and 1 Gy/h and a parabolic relationship at dose rates between 0.5 and 2.6 Gy/h.

Apoptosis↗

PubMed2003

A bioluminescent sensor for high throughput toxicity classification.

A high throughput toxicity monitoring and classification biosensor system has been successfully developed using four immobilized bioluminescent Escherichia coli strains, DPD2511, DPD2540, DPD2794 and TV1061, which have plasmids bearing a fusion of a specific promoter to the luxCDABE operon. The bioluminescence of DPD2511 increases in the presence of oxidative damage, DPD2540 by membrane damage, DPD2794 by DNA damage and TV1061 by protein damage. In the developed biosensor these strains are immobilized in a single 96 well plate using an LB-agar matrix, and are able to detect the toxicities of hydrogen peroxide, phenol and mitomycin C in water samples. As the concentration of each chemical was increased, the bioluminescence levels from the corresponding wells, containing either DPD2511, DPD2540, DPD2794 or TV1061, increased. This increase in bioluminescence followed a dose dependent response to the toxic chemicals within a specific concentration range. In particular, each test requires only 4 h to give clear bioluminescent response signature. Storage of the biosensor at 4 degrees C for 2 weeks caused no change in its dose-dependent response. The fast and easy detection of oxidative, membrane, protein and DNA damaging agents in aqueous environments is possible due to the high throughput capability of this biosensor.

Biosensing Techniques↗

PubMed2003

Effects of endocrine disrupting chemicals on distinct expression patterns of estrogen receptor, cytochrome P450 aromatase and p53 genes in oryzias latipes liver.

Expression pattern analysis of three genes, i.e., the estrogen receptor (ER), cytochrome P450 aromatase (CYP19), and p53 genes, in Japanese Medaka liver was studied in the presence of 17beta-estradiol, nonylphenol, and bisphenol A. Using the distinct expression patterns of these three genes, the different transcriptional mechanisms by endocrine disrupting chemicals (EDCs), and the stability of the transcripts of each gene, were examined. In tests done with both male and female Medaka over 10 days, it was found that each gene had a specific expression pattern. The expression of the ER gene increased rapidly for the first 2 days and then leveled out and maintained a stable expression level, while the CYP19 gene expression showed a fairly consistent increase in the expression levels after exposure. In contrast to both these genes, the p53 gene expression levels reached a maximum value within 2 days and then gradually decreased for the remainder of the experiment. These findings suggest that different expression mechanisms may exist for these genes in Japanese Medaka in response to the presence of EDCs.

Animals↗

PubMed2003

Degradation of 2,4,6-trinitrotoluene by immobilized horseradish peroxidase and electrogenerated peroxide.

This paper presents horseradish peroxidase (HRP)-catalyzed removal of 2,4,6-trinitrotoluene (TNT) by an electrochemical packed-bed flow reactor operated in a circulating batch mode with the help of in situ generated hydrogen peroxide. HRP immobilized on the reticulated vitreous carbon electrode was prepared for the cyclic voltammetry of 2,4,6-TNT. Effects of pH and temperature on the TNT electroreduction in 0.2M phosphate buffer saturated with oxygen were examined. HRP immobilized carbon electrode was capable of catalyzing the oxidation and detoxification of 44 microM TNT in aqueous solution under optimized conditions. The removal rate of TNT for the electroenzymatic method was much greater than for electrochemical and biochemical methods. Stoichiometric and kinetic studies indicated that the hydrogen peroxide was utilized more effectively in the electroenzymatic method. Denitrification as intermediate reaction was also investigated.

Electrochemistry↗

PubMed2003

Evaluation of a high throughput toxicity biosensor and comparison with a Daphnia magna bioassay.

A high throughput toxicity biosensor has been designed and constructed using recombinant Escherichia coli cells, containing stress specific promoters (recA, fabA, or katG) or constitutive promoters (lac) fused to luciferase genes originating from Vibrio fisheri. These genetically engineered cells were immobilized in 96 well plates. By optimizing cell immobilization conditions and the strains' response specificity to toxic chemicals, bioluminescent outputs decreased or increased dose-dependently upon adding test chemicals. However, to date the toxicity data obtained using this biosensor have not been compared with the results of other toxicity tests. Phenolics were chosen to evaluate the correlation between the LD50 and the EC50 (GC2) or EC120 (DPD2540) of Daphnia magna and E. coli, respectively. Toxicity data obtained from constitutive strains by bioluminescent level decrements were compared with the results from D. magna as a standard. LD50 values were used as parameters of D. magna toxicity and EC50 of EC120 values were used for the immobilized biosensor. In the DPD2540 test, phenolics, membrane damaging toxic chemicals, for testing immobilized stress specific bacterial strains trigger dose-dependant bioluminescence increase within specific concentration. Although the stress specific responsiveness from the strains could not be compared with D. magna's LD50 values, these responses offer additional information, such as upon the mode of toxic action in the sample, in addition to the cellular toxicity results as indicated by the EC50. This novel high throughput toxicity biosensor can be implemented to investigate the toxicity of any other soluble materials, and can be used as a standardization tool for the evaluation of toxicity.

Animals↗

PubMed2003

Monitoring and classification of PAH toxicity using an immobilized bioluminescent bacteria.

An immobilized recombinant bioluminescent Escherichia coli strain, harboring a lac::luxCDABE fused plasmid, which shows lower bioluminescence levels when cellular metabolism is inhibited, was used to monitor the cellular toxicity of polycyclic aromatic hydrocarbons (PAHs). PAHs, classified as pericondensed (PCPAHs) or catacondensed (CCPAHs) according to their molecular structures, were differentiable according to the response of this biosensor. Only CCPAHs were found to cause cellular toxicity, resulting in a dose-dependent decrease in the bioluminescent output. The induction of cellular toxicity by CCPAHs and PCPAHs was compared with acute toxicity predictions obtained using the quantitative structure-activity relationship (QSAR) model. A good relationship was obtained between the toxicities determined with the bioluminescent response of the immobilized bacterium GC2 and the QSAR model. It was also found that the present study offers a new method of predicting the cellular toxicities of CCPAHs or PCPAHs using this biosensor.

Biosensing Techniques↗