Biomedical subjects

Man Bock Gu

Publications and source records attributed to Man Bock Gu.

33 records · Page 2Linked to original sources

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↗

PubMed2003

Bacterial detection of the toxicity of dioxins, polychlorinated diphenyls, and polybrominated diphenyl ethers.

Polychlorinated dibenzo-p-dioxins (PCDDs), biphenyls (PCBs), and polybrominated diphenyl ethers (PBDEs) were found to induce several specific stresses within bacterial cells. Four different recombinant Escherichia coli with specific stress promoters (i.e., the recA responsive to DNA damage, fabA responsive to membrane damage, katG responsive to oxidative damage, and grpE responsive to protein damage) that were fused to the lux operon from Vibrio fischeri showed very unique specificities in terms of their stress responses in the presence of PCDD and PCBs. In addition, a recombinant bacterium with the lac promoter fused to the lux operon from Xenorhabdus luminescence also showed dose-dependent responses via a loss of bioluminescence because of the addition of the PCDDs and PCBs. Brominated diphenyl ethers (BDE) -47, -99, and -153 congeners, however, were not found to induce any stress within the bacterial cells, indicating that these chemicals do not stimulate any cellular stresses related to those tested. These three congeners, however, did result in different levels of general cellular toxicity, which was found to be dependent on the position of the bromine. Finally, the cellular toxicity within the bacteria was found to increase when exposed to mixtures of dioxins, PCBs, and PBDEs, possibly from synergistic effects.

DNA Damage↗

PubMed2003

Specific responses of bacterial cells to dioxins.

Five different recombinant bioluminescent strains of Escherichia coli that contain the recA (responsive to DNA damage related stress), fabA (membrane damage), katG (oxidative damage), grpE (protein damage), and lac (constitutive expression, general toxicity) promoters fused to the bacterial lux operon from either Vibrio fischeri or Photorhabdus luminescens were used to describe the different mechanisms of toxicity that several dibenzo-p-dioxins and dibenzofurans have on bacteria, as well as to determine whether bacteria can sensitively detect the presence of these compounds. 2,3,7,8-Tetrachlorodibenzo-p-dioxin (2,3,7,8-TCDD) was found to cause only DNA-related damage to bacterial cells. However, the four stress-responsive strains showed positive responses after addition of 1,2,3,4-tetrachlorodibenzo-p-dioxin (1,2,3,4-TCDD), whereas 2,3,7,8-tetrachlorodibenzo-p-furan (2,3,7,8-TCDF) caused only DNA. oxidative, and protein damage. However, 2,8-dichlorodibenzo-p-dioxin (2,8-DCDD) was not found to induce any stresses tested for in this study, that is, DNA, membrane, oxidative, and protein damage, indicating that each congener might differentially interact with the cell, stimulating differential stress responses within them. By using the constitutive strain, we found that the level of cellular toxicity experienced due to the addition of these four dioxins decreased in the order of 2,3,7,8-TCDD (the most toxic). 1,2,3,4-TCDD, 2.8-DCDD, and 2,3,7,8-TCDF. The 20% effective concentration (EC20), defined in this study the concentration of chemical that causes a 20% decrease in the bioluminescence 60 min after induction, was only 0.1 microg/L for 2,3,7,8-TCDD, a value that is lower than that of the other congeners and demonstrates that 2,3,7,8-TCDD was the most toxic compound tested in this study.

Bacterial Proteins↗

PubMed2002

Enhancing the sensitivity of a two-stage continuous toxicity monitoring system through the manipulation of the dilution rate.

Optimization of the dilution rates has been studied to provide an enhanced sensitivity to toxicity by several recombinant bioluminescent Escherichia coli strains, TV1061 (grpE::luxCDABE), DPD2794 (recA::luxCDABE) and DPD2540 (fabA::luxCDABE), in the two-stage continuous toxicity monitoring system. It was found that the sensitivity of both TV1061 and DPD2794 to a pulse injection of phenol and mitomycin C increased with a decrease in the dilution rate. The sensitivity, however, for all the strains to step injections of the toxic chemicals was found to increase with an increase in the dilution rate up to a certain dilution rate and then decreased, mainly due to the rapid washing out of the injected chemicals. The response kinetics of the strains were explained by evaluating the mode of action of the recombinant bioluminescent bacteria to toxicity with the dilution rate, the operating parameter of minibioreactors under consideration in this study.

Bacterial Proteins↗

PubMed2002

Toxicity monitoring and classification of endocrine disrupting chemicals (EDCs) using recombinant bioluminescent bacteria.

A recombinant bioluminescent Escherichia coli, DPD2794, containing the recA promoter region fused to luxCDABE originating from Vibrio fischeri was used for detecting genotoxicity caused by endocrine disrupting chemicals (EDCs) to cells. As well, several other recombinant bioluminescent bacteria, including TV1061, which is sensitive to protein damage (grpE::luxCDA BE), DPD2511, sensitive to oxidative damage (katG::luxCDABE), and DPD2540, sensitive to membrane damage (fabA::luxCDABE), were used for evaluating other possible modes of toxicity. It was found that the recombinant bacteria could monitor the toxic effect, not estrogenic effect, due to the presence of various EDCs through the measurement of bioluminescence (BL) and cell growth rate, which depend upon the type of toxicity occurring and which of the four strains was used. In addition, it was found that the damage caused by EDCs can be classified into several groups upon their mechanisms of toxic action.

Bacterial Proteins↗

PubMed2002

A portable toxicity biosensor using freeze-dried recombinant bioluminescent bacteria.

A portable biosensor has been developed to meet the demands of field toxicity analysis. This biosensor consists of three parts, a freeze-dried biosensing strain within a vial, a small light-proof test chamber, and an optic-fiber connected between the sample chamber and a luminometer. Various genetically engineered bioluminescent bacteria were freeze-dried to measure different types of toxicity based upon their modes of action. GC2 (lac::luxCDABE), a constitutively bioluminescent strain, was used to monitor the general toxicity of samples through a decrease in its bioluminescence, while specific toxicity was detected through the use of strains such as DPD2540 (fabA::luxCDABE), TV1061 (grpE::luxCDABE), DPD2794 (recA::luxCDABE), and DPD2511 (katG::luxCDABE). These inducible strains show an increase in bioluminescence under specific stressful conditions, i.e. membrane-, protein-, DNA-, and oxidative-stress, respectively. The toxicity of a sample could be detected by measuring the bioluminescence 30 min after addition to the freeze-dried strains. In an attempt to enhance the sensitivity of the freeze-dried cells, glucose and Tween 80 were tested as additives. It was found that the addition of glucose had a negative effect on the viability of the freeze-dried cells, while samples having Tween 80 showed an increase in their viability. On the other hand, the addition of either Tween 80 or glucose decreased the final bioluminescent response of DPD2540 in response to 4-chlorophenol. Using these strains, many different chemicals were tested and characterized. This portable biosensor, with a very simple protocol, can be used for field sample analysis and the monitoring of various water systems on-site.

Biosensing Techniques↗

PubMed2002

Enhancement in the sensitivity of a gas biosensor by using an advanced immobilization of a recombinant bioluminescent bacterium.

A genetically engineered bioluminescent bacterium (lac::luxCDABE) was immobilized to develop a whole cell biosensor for the detection of toxic gaseous chemicals. The toxicity of chemicals can be evaluated through the bioluminescent reaction as it reduces in intensity when the cells experience toxic or lethal conditions. This whole cell biosensor was fabricated, using an immobilization technique utilizing solid agar medium, for the measurement of toxicity through direct contact of the cells with the gas. To enhance the sensitivity of the biosenor, glass beads were used and the thickness of the agar layer was reduced. The bioluminescent response was measured using a fiber optic probe connected between the biosensor kit and a luminometer. As sample gaseous toxic chemicals, BTEX (Benzene, Toluene, Ethylbenzene, and Xylene) gases were selected and their vapors were produced by a gas generation system. The concentrations of the gaseous chemicals injected into the chamber were controlled by the time of exposure and were measured using a portable gas chromatograph (Allstech., USA). Additions of glass beads facilitated gas diffusion through the solid medium, making the biosensor more sensitive. In addition, a thinner matrix layer was more advantageous for the detection of gas toxicity.

Bacteria↗

PubMed2002

Enhanced degradation of an endocrine-disrupting chemical, butyl benzyl phthalate, by Fusarium oxysporum f. sp. pisi cutinase.

Compared to yeast esterase, fungal cutinase degraded butyl benzyl phthalate (BBP) far more efficiently; i.e., almost 60% of the BBP disappeared within 7.5 h. Also, the final chemical composition significantly depended on the enzyme used. Toxicity monitoring using bioluminescent bacteria showed that butyl methyl phthalate, a major product of degradation by esterase, was an oxidative toxic hazard.

Carboxylic Ester Hydrolases↗