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Biomedical subjects

X C Le

Publications and source records attributed to X C Le.

At least 19 recordsLinked to original sources
PubMed2001

Detection of human immunodeficiency virus type 1 reverse transcriptase using aptamers as probes in affinity capillary electrophoresis.

An affinity capillary electrophoresis/laser-induced fluorescence (CE/LIF) assay was developed for direct and specific detection of reverse transcriptase (RI) of the type 1 human immunodeficiency virus (HIV-1) using fluorescently labeled single-stranded DNA aptamers as probes. The aptamer used (RT 26) is specific for HIV-1 RT, and it exhibited no cross-reactivity with RTs of the enhanced avian myeloblastosis virus (AMV), the Moloney murine leukemia virus (MMLV), or denatured HIV-1 RT. An affinity complex of RT 26-HIV-1 RT was readily formed, and calibration curves were linear up to 50 nM (6 microg/mL) HIV-1 RT concentration, with both the free probe and complex peak usable for analytical quantitation. Cell culture media (RPMI with 10% fetal bovine serum) interfered with the assay and aptamer-HIV-1 RT binding. Nonspecific binding was observed in low or undiluted culture, necessitating at least 100-fold dilution for analysis of raw culture samples.

Calibration↗
PubMed2001

Analyses of micronuclei in exfoliated epithelial cells from individuals chronically exposed to arsenic via drinking water in inner Mongolia, China.

The groundwater in Bayingnormen (Ba Men), located in Central West Inner Mongolia, China, is naturally contaminated with arsenic at concentrations ranging from 50 microg/L to 1.8 mg/L. Various adverse health effects in this region, including cancer, have been linked to arsenic exposure via drinking water. A pilot study was undertaken to evaluate frequencies of micronuclei (MN), as measures of chromosomal alterations, in multiple exfoliated epithelial cell types from residents of Ba Men chronically exposed to arsenic via drinking water. Buccal mucosal cells, airway epithelial cells in sputum, and bladder urothelial cells were collected from 19 residents exposed to high levels of arsenic in drinking water (527.5 +/- 24 microg/l), and from 13 control residents exposed to relatively low levels of arsenic in drinking water (4.4 +/- microg/L). Analytical results from these individuals revealed that MN frequencies in the high-exposure group were significantly elevated to 3.4-fold over control levels for buccal and sputum cells, and to 2.7-fold over control for bladder cells (increases in MN frequency significant at p < .001 for buccal cells; p < .01 for sputum cells; p < .05 for bladder cells). When smokers were excluded from high-exposure and control groups the effects of arsenic were observed to be greater, although only in buccal and sputum cells; approximately 6-fold increases in MN frequency occurred in these tissues. The results indicate that residents of Ba Men chronically exposed to high levels of arsenic in drinking water reveal evidence of genotoxicity in multiple epithelial cell types; higher levels of induced MN were observed in buccal and sputum cells than in bladder cells.

Adult↗
PubMed2001

Detection of DNA adducts of benzo[a]pyrene using immunoelectrophoresis with laser-induced fluorescence. Analysis of A549 cells.

Detection of benzo[a]pyrene diol epoxide (BPDE)-damaged DNA in a human lung carcinoma cell line (A549) has been performed using free zone affinity capillary electrophoresis with laser-induced fluorescence (LIF). Using BPDE as a model carcinogenic compound, the speed, sensitivity and specificity of this technique was demonstrated. Under free zone conditions, an antibody bound adduct was baseline-resolved from an unbound adduct in less than 2 min. The efficiencies of separation were in excess of 6 x 10(5) and 1 x 10(6) plates per meter for the antibody-bound and unbound adducts, respectively. Separation using a low ionic strength buffer permitted the use of a high electric field (830 V/cm) without the loss of resolving power. Using LIF detection, a concentration detection limit of roughly 3 x 10(-10) M was achieved for a 90-mer oligonuleotide containing a single BDPE. The use of formamide in the incubation buffer to enhance denaturing of DNA did not affect the stability of the complex between the antibody and the adducts. Using a fluorescently labeled BPDE-modified DNA adduct probe, a competitive assay was established to determine the levels of BPDE-DNA adducts in A549 cells.

7,8-Dihydro-7,8-dihydroxybenzo(a)pyrene 9,10-oxide↗
PubMed2001

Factors influencing the removal of thymine glycol from DNA in gamma-irradiated human cells.

The toxic and mutagenic effects of ionizing radiation are believed to be caused by damage to cellular DNA. We have made use of a novel immunoassay for thymine glycol to examine the removal of this lesion from the DNA of irradiated human cells. Because of the sensitivity of the assay, we have been able to keep the radiation doses at or below the standard clinical dose of 2 Gy. Our initial observations indicated that although removal of thymine glycol is > 80% complete by 4 h post-irradiation with 2 Gy, there is a lag of 30-60 min before repair commences. However, if cells are irradiated with 0.25 Gy 4 h prior to the 2-Gy dose, removal of the thymine glycols commences immediately after the second irradiation, suggesting that repair of thymine glycol is inducible. Our current studies are directed at two aspects of the repair process, (1) factors involved in the repair process leading up to and including glycosylase-mediated removal of thymine glycol and (2) the control of the inducible response. We have observed that mutation of the XPG gene drastically reduced the level and rate of global removal of thymine glycol (induced by 2-Gy irradiation), and there was no evidence for an inducible response. Similar results were seen with a Cockayne syndrome B (CSB) cell line. We have also examined repair in quiescent and phytohemagglutinin-stimulated human lymphocytes. Both show similar kinetics for the rate of removal of thymine glycol under induced and noninduced conditions.

Animals↗
PubMed2001

Synthesis, characterization, and applications of a fluorescent probe of DNA damage.

We have designed and generated a 90-mer oligonucleotide that contains a single adduct of benzo[a]pyrene diol epoxide (BPDE) and that is fluorescently labeled. The known amount of BPDE adduct in a given length of DNA makes this probe a useful standard for DNA damage assay. The BPDE-90-mer was fluorescently labeled with tetramethylrhodamine to allow for high sensitivity detection with laser-induced fluorescence (LIF). The binding of both double-stranded and single-stranded BPDE-90-mer with three anti-BPDE antibodies was studied using affinity capillary electrophoresis (CE). Formation of antibody complex with BPDE-90-mer results in a shift in migration time from that of the unbound BPDE-90-mer. Affinity CE/LIF studies suggest that antibody 8E11 has high-affinity suitable for immunoassay of BPDE-DNA adducts. A competitive immunoassay using the fluorescent probe and CE/LIF is demonstrated for the analysis of BPDE-DNA adducts in A549 human lung carcinoma cells incubated with 2.5, 5, and 10 microM BPDE for 2 h. The design of the 90-mer probe is flexible to substitute different DNA damage types with relative ease. The fluorescent 90-mer is composed of six shorter oligonucleotides. The sequence of the two center oligonucleotides may be changed depending on the desired DNA lesion measurement. By inserting different damaged oligonucleotides, a variety of DNA damage systems can be investigated using the same CE/LIF approach.

Antibodies↗
PubMed2001

Speciation of arsenic using solid phase extraction cartridges.

Various solid phase extraction (SPE) cartridges were investigated for speciation of arsenite [As(III)], arsenate [As(v)], monomethylarsonic acid (MMA) and dimethylarsinic acid (DMA). Cartridges containing different types of sorbent materials were tested for arsenic retention and elution characteristics. Alumina cartridges were found to completely retain all the four target arsenic species, and are suitable for removal and preconcentration purposes. For speciation analysis, different arsenic species were separated on the basis of their selective retention on and elution from specific cartridges. DMA was retained on a resin-based strong cation exchange cartridge and eluted with 1.0 M HCl. MMA and As(v) were both retained on a silica-based strong anion exchange cartridge and sequentially eluted with 60 mM acetic acid (for MMA) and 1.0 M HCl [for As(v)]. As(III) was not retained on either cartridge and remained in solution. Arsenic species in solution and those eluted from the cartridges were subsequently quantified by using flow injection with hydride generation atomic fluorescence spectrometry (FI-HGAFS) and hydride generation atomic absorption spectrometry (FI-HGAAS). A detection limit of 0.05 microg L(-1) arsenic in water sample was achieved using HGAFS. An application of the method was demonstrated at a drinking water treatment facility. As(III) and As(v) species were determined in water at various stages of treatment. The method is suitable for routine determination of trace levels of arsenic in drinking water to comply with more stringent environmental regulations.

Adsorption↗
PubMed2000

Studies of protein--DNA interactions by capillary electrophoresis/laser-induced fluorescence polarization.

Protein-DNA interactions were studied on the basis of capillary electrophoretic separation of bound from free fluorescent probe followed by on-line detection with laser-induced fluorescence polarization. Changes in electrophoretic mobility and fluorescence anisotropy upon complex formation were monitored for the determination of binding affinity and stoichiometry. The method was applied to study the interactions of single-stranded DNA binding protein (SSB) with synthetic oligonucleotides and single-stranded DNA. Increases in fluorescence anisotropy and decreases in electrophoretic mobility upon their binding to SSB were observed for the fluorescently labeled 11-mer and 37-mer oligonucleotide probes. Fluorescence anisotropy and electrophoretic mobility were used to determine the binding constants of the SSB with the 11-mer (5 x 10(6) M(-1)) and the 37-mer (23 x 10(6) M(-1)). Alternatively, a fluorescently labeled SSB was used as a probe, and the formation of multiple protein-DNA complexes that differ in stoichiometry was observed. The results demonstrate the applicability of the method to study complex interactions between protein and DNA.

Base Sequence↗
PubMed2000

Speciation of key arsenic metabolic intermediates in human urine.

Biomethylation is the major human metabolic pathway for inorganic arsenic, and the speciation of arsenic metabolites is essential to a better understanding of arsenic metabolism and health effects. Here we describe a technique for the speciation of arsenic in human urine and demonstrate its application to the discovery of key arsenic metabolic intermediates, monomethylarsonous acid (MMAIII) and dimethylarsinous acid (DMAIII), in human urine. The study provides a direct evidence in support of the proposed arsenic methylation pathway in the human. The finding of MMAIII and DMAIII in human urine, along with recent studies showing the high toxicity of these arsenicals, suggests that the usual belief of arsenic detoxification by methylation needs to be reconsidered. The arsenic speciation technique is based on ion pair chromatographic separation of arsenic species on a 3-micron particle size column at 50 degrees C followed by hydride generation atomic fluorescence detection. Speciation of MMAIII, DMAIII, arsenite (AsIII), arsenate (AsV), monomethylarsonic acid (MMAV), and dimethylarsinic acid (DMAV) in urine samples is complete in 6 min with detection limits of 0.5-2 micrograms/L. There is no need for any sample pretreatment. The capability of rapid analysis of trace levels of arsenic species, which resulted in the findings of the key metabolic intermediates, makes the technique useful for routine arsenic speciation analysis required for toxicological and epidemiological studies.

Arsenic↗
PubMed2000

DMPS-arsenic challenge test. II. Modulation of arsenic species, including monomethylarsonous acid (MMA(III)), excreted in human urine.

The administration of sodium 2,3-dimercapto-1-propane sulfonate (DMPS) to humans chronically exposed to inorganic arsenic in their drinking water resulted in the increased urinary excretion of arsenic, the appearance and identification of monomethylarsonous acid (MMA(III)) in their urine, and a large decrease in the concentration and percentage of urinary dimethylarsinic acid (DMA). This is the first time that MMA(III) has been detected in the urine. In vitro biochemical experiments were then designed and performed to understand the urinary appearance of MMA(III) and decrease of DMA. The DMPS-MMA(III) complex was not active as a substrate for the MMA(III) methyltransferase. The experimental results support the hypothesis that DMPS competes with endogenous ligands for MMA(III), forming a DMPS-MMA complex that is readily excreted in the urine and points out the need for studying the biochemical toxicology of MMA(III). It should be emphasized that MMA(III) was excreted in the urine only after DMPS administration. The results of these studies raise many questions about the potential central role of MMA(III) in the toxicity of inorganic arsenic and to the potential involvement of MMA(III) in the little-understood etiology of hyperkeratosis, hyperpigmentation, and cancer that can result from chronic inorganic arsenic exposure.

Adult↗
PubMed2000

Measuring DNA damage using capillary electrophoresis with laser-induced fluorescence detection.

Damage to cellular DNA is implicated in the early stages of carcinogenesis and in the cytotoxicity of many anticancer agents, including ionizing radiation. Sensitive techniques are required for measuring cellular levels of DNA damage. We describe in detail a novel immunoassay that makes use of the resolving power of capillary electrophoresis and the sensitivity of laser-induced fluorescence detection. An example is given of the detection of thymine glycol in DNA produced by irradiation of human cells with a clinical dose of 2 Gy. A detection limit of approximately 10(-21) mol allowed us to monitor the repair of the lesion and to suggest that the cellular repair response may be inducible.

Animals↗
PubMed2000

Occurrence of monomethylarsonous acid in urine of humans exposed to inorganic arsenic.

Monomethylarsonous acid (MMA(III)) has been detected for the first time in the urine of some humans exposed to inorganic arsenic in their drinking water. Our experiments have dealt with subjects in Romania who have been exposed to 2.8, 29, 84, or 161 microg of As/L in their drinking water. In the latter two groups, MMA(III) was 11 and 7% of the urinary arsenic while the monomethylarsonic acid (MMA(V)) was 14 and 13%, respectively. Of our 58 subjects, 17% had MMA(III) in their urine. MMA(III) was not found in urine of any members of the group with the lowest level of As exposure. If the lowest-level As exposure group is excluded, 23% of our subjects had MMA(III) in their urine. Our results indicate that (a) future studies concerning urinary arsenic profiles of arsenic-exposed humans must determine MMA(III) concentrations, (b) previous studies of urinary profiles dealing with humans exposed to arsenic need to be re-examined and re-evaluated, and (c) since MMA(III) is more toxic than inorganic arsenite, a re-examination is needed of the two hypotheses which hold that methylation is a detoxication process for inorganic arsenite and that inorganic arsenite is the major cause of the toxicity and carcinogenicity of inorganic arsenic.

Adolescent↗
PubMed2000

Determination of monomethylarsonous acid, a key arsenic methylation intermediate, in human urine.

In this study we report on the finding of monomethylarsonous acid [MMA(III)] in human urine. This newly identified arsenic species is a key intermediate in the metabolic pathway of arsenic biomethylation, which involves stepwise reduction of pentavalent to trivalent arsenic species followed by oxidative addition of a methyl group. Arsenic speciation was carried out using ion-pair chromatographic separation of arsenic compounds with hydride generation atomic fluorescence spectrometry detection. Speciation of the inorganic arsenite [As(III)], inorganic arsenate [As(V)], monomethylarsonic acid [MMA(V)], dimethylarsinic acid [DMA(V)], and MMA(III) in a urine sample was complete in 5 min. Urine samples collected from humans before and after a single oral administration of 300 mg sodium 2,3-dimercapto-1-propane sulfonate (DMPS) were analyzed for arsenic species. MMA(III) was found in 51 out of 123 urine samples collected from 41 people in inner Mongolia 0-6 hr after the administration of DMPS. MMA(III )in urine samples did not arise from the reduction of MMA(V) by DMPS. DMPS probably assisted the release of MMA(III) that was formed in the body. Along with the presence of MMA(III), there was an increase in the relative concentration of MMA(V) and a decrease in DMA(V) in the urine samples collected after the DMPS ingestion.

Arsenic↗
PubMed1999

Capillary electrophoretic immunoassays for digoxin and gentamicin with laser-induced fluorescence polarization detection.

New immunoassays for therapeutic drugs digoxin and gentamicin have been described, which involved the separation of free and antibody-bound drug by capillary electrophoresis (CE) and the detection by laser-induced fluorescence polarization (LIFP). While the fluorescein-labeled digoxin and gentamicin (tracers) displayed negligible fluorescence polarization in solution, the complex formation between these small molecules and their antibodies resulted in substantial increases in fluorescence polarization due to the increase in molecular size. The LIFP detection, capable of measuring vertically and horizontally polarized fluorescence components simultaneously, provides enhanced capability for the identification of complex in capillary electrophoretic immunoassays. Proper adjustments of the running buffer pH and the ratio of antibody to tracer are essential for optimization of the performance of these assays. The digoxin-antibody complex remained stable during CE separation with running buffer pH ranging from 9.3 to 12. Calibration curves covering a concentration range of 0.05 to 0.5 ng/ml were obtained with a running buffer of pH 12. The concentration and mass detection limits were 0.02 ng/ml and 26 zmol, respectively. For gentamicin assay, the running buffer pH 10 was used to reduce the adsorption of the tracer while minimizing the dissociation of the antibody-tracer complex during the separation. The calibration curves covered a concentration range 0.05-1.0 microg/ml, with a concentration detection limit of 25 ng/ml and a mass detection limit of 52 amol of gentamicin.

Anti-Arrhythmia Agents↗
PubMed1999

Fluorescence polarization studies of affinity interactions in capillary electrophoresis.

Capillary electrophoresis (CE) combined with molecular recognition for ultrasensitive bioanalytical applications often requires the formation of stable complexes between an analyte and its binding partner. Previous studies of binding interactions using CE involve multiple-step titration experiments and are time-consuming. We describe a simple method based on laser-induced fluorescence polarization (LIFP) detection for CE separation, which allows for on-line monitoring of affinity complex formation. Because fluorescence polarization is sensitive to changes in the rotational diffusion arising from molecular association or dissociation, it is capable of providing information on the formation of affinity complexes prior to or during CE separation. Applications of the CE/LIFP method to three binding systems including vancomycin and its antibody, staphylococcal enterotoxin A and its antibody, and trp operator and trp repressor were demonstrated, representing peptide-protein, protein-protein, and DNA-protein interactions. The affinity complexes were readily distinguished from the unbound molecules on the basis of their fluorescence polarization. The relative increase in fluorescence polarization upon complex formation varied with the molecular size of the binding pairs.

Antibodies↗
PubMed1999

Competitive immunoassay for staphylococcal enterotoxin A using capillary electrophoresis with laser-induced fluorescence detection.

Staphylococcal enterotoxins are a family of toxic proteins secreted by S. aureus. Using capillary electrophoresis (CE) linked with laser-induced fluorescence, a highly sensitive and selective assay using antibody-antigen recognition was developed for the determination of Staphylococcal enterotoxin A. Staphylococcal enterotoxin A (SEA) was chemically labeled with fluorescein and the product was used as a fluorescent tracer. A competitive assay was developed to detect SEA at concentrations between 0.3 nM and 6.5 nM with standard deviations of less than 5%. The detection limit was found to be 3 amol with the potential improvement by further optimization of the assay. No cross-reactivity between staphylococcal enterotoxin B and the SEA antibody was found at the concentrations used for the CE immunoassay.

Electrophoresis, Capillary↗
PubMed1999

Capillary electrophoresis coupled with laser-induced fluorescence polarization as a hybrid approach to ultrasensitive immunoassays.

Immunoassays using capillary electrophoresis with laser-induced fluorescence detection (CE-LIF) is a powerful approach to the determination of trace amounts of analytes in a complex biological matrix. However, its applicability is limited by the requirement that the free and bound tracer (fluorescently labeled compound) be resolved for their identification and quantitation. Here we show that replacing LIF with laser-induced fluorescence polarization (LIFP) permits ultrasensitive immunoassays to be performed with or without the separation of the free and bound tracer. A binding system involving cyclosporin A (CyA) and monoclonal antibody to CyA was chosen to demonstrate both homogeneous and heterogeneous immunoassay approaches. In the homogeneous scheme where the free and bound tracer were not separated, the fluorescence polarization of the mixture was a quantitative measure of the antibody-bound tracer. The concentration and mass detection limits for CyA using the homogeneous competitive assay were found to be 1 nM and 1 amol (10(-18) mol), respectively. The heterogeneous assay involved a nearly baseline separation of the free and bound tracer using CE with a phosphate running buffer of pH 7.0. The complex of the tracer with the antibody had a fluorescence polarization of approximately 0.24 whereas the free tracer had negligible polarization. The fluorescence polarization was independent of analyte concentration, and the fluorescence intensity of either the free or bound tracer was used for quantitation. Results from both assays suggest that the CE-LIFP approaches may have a wider application than the immunoassays based on either CE-LIF or fluorescence polarization alone.

Antibodies, Monoclonal↗
PubMed1999

Zinc exposure in Chinese foundry workers.

BACKGROUND: Inhalational exposure to zinc oxide fumes is associated with metal fume fever, a self-limited but very uncomfortable condition closely resembling influenza. Very little is known regarding the toxicokinetics of inhaled zinc, making the interpretation of zinc measurements in serum and urine problematic. METHODS: Twenty workers in a zinc foundry in Baiyin, Peoples' Republic of China, were investigated with serial examinations by a physician, chest radiographs, and spirometry. Exposure assessment consisted of the measurement of zinc in serum, urine, and personal air samples. RESULTS: No cases of metal fume fever were observed during the study period despite exposures to as high as 36.3 mg/m3 over less than 4 hr. In addition, no radiographic or functional changes were noted. Serum zinc levels of all workers were within the reference range and did not correlate with external exposure measurements. However, elevations were noted in urinary zinc levels, which showed a significant association (Spearman's correlation coefficient = 0.47, P = 0.04) between exposure to zinc and urine zinc. CONCLUSIONS: These results provide exposure measurements for zinc at which workers demonstrate tolerance to the development of metal fume fever. Furthermore, they suggest that urine may be the preferred biological medium for the assessment of zinc exposure.

Adult↗