Biomedical subjects

D P Hsieh

Publications and source records attributed to D P Hsieh.

At least 19 recordsLinked to original sources

PubMed2001

Purification and in vitro folding of recombinant human thrombopoietin receptor expressed in Escherichia coli.

Thrombopoietin receptor (TPOR) is a member of the cytokine receptor superfamily expressed primarily on hematopoietic cells. TPOR plays an important role in regulating platelet production. Due to its low expression level in human tissue, studies on the biochemical and biophysical properties of TPOR have been limited. In the present study, an extracellular domain of recombinant human TPOR (rh TPOR-EN) was expressed in Escherichia coli as inclusion bodies. Purification was achieved by metal chelated chromatography under denaturing condition and was refolded by gel filtration chromatography. Far UV circular dichroism spectroscopy and surface plasmon resonance experiments were performed to demonstrate that the protein was in a refolded state and could bind with its ligand. Thus, a production and purification scheme was developed by which sufficient quantities of rh TPOR-EN can be made available for biochemical and biophysical characterizations.

Binding Sites↗

PubMed2001

Dynamics of C2 toxin and chlorophyll-a formation in the dinoflagellate Alexandrium tamarense during large scale cultivation.

The production of paralytic shellfish toxins (PSTs) by the dinoflagellate Alexandrium tamarense ATCI01, a toxigenic strain isolated from South China coastal waters, was studied in batch cultures in relatively large volumes (20l). Under nutrient-replete conditions, this strain produced C2 toxin (C2T) as a predominant PST. In a 15-day production culture, phosphate was depleted by day 4, the stationary phase began at day 6, and the toxin productivity peaked at day 10, in which the cell content of C2T reached 76 fmol per cell. Much of the toxin was produced after the depletion of phosphate in the medium suggesting that C2T is a secondary metabolite. Aeration with small bubbles was useful in increasing cell mass and toxin yield. Chlorophyll-a (Chl-a) was formed in algal cells until the culture entered the stationary phase, after which Chl-a began to disappear rapidly from the culture while the C2T content continued to rise. These results suggest a metabolic relationship between Chl-a and C2T.

Air↗

PubMed2001

Laboratory bioproduction of paralytic shellfish toxins in dinoflagellates.

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Animals↗

PubMed2001

Influences of metal concentration in phytoplankton and seawater on metal assimilation and elimination in marine copepods.

Radiotracer experiments were conducted to examine the influence of the concentration of Cd, Se, and Zn in ingested phytoplankton (dinoflagellate Prorocentrum minimum and diatom Thalassiosira weissflogii) and in ambient seawater on metal assimilation and elimination efficiencies of three marine copepods, Acartia spinicauda, Paracalanus aculeatus, and Calanus sinicus. The assimilation efficiencies (AEs) decreased by 1.7 to 2.0 times, 1.4 to 4.1 times, and 1.3 to 2.2 times in the copepods with an increase in metal concentration in ingested algae by 16 to 84 times, 14 times, and 45 to 153 times, for Cd, Se, and Zn, respectively. However, the physiologic turnover rate constant was relatively independent of the metal concentration in copepods. No evidence was found of any interaction between Cd and Zn in their assimilation by copepods. Assimilation efficiencies of Cd were higher in copepods feeding on the dinoflagellate P. minimum, whereas the AEs of Zn were higher in copepods feeding on the diatom T. weissflogii. Differences in metal distribution in algal cytoplasm at different ambient metal concentrations may be partially responsible for the observed influence of metal concentration in algal cells on metal assimilation in copepods. However, metal desorption within the gut of the copepod may have little influence on metal assimilation, as a result of the short gut residence time of food particles and the neutral gut pH. Our study also indicated that the ingestion rate of copepods was reduced by a higher concentration of Cd and Se, but was not affected by Zn concentration in the food particles. Consequently, partial regulation of metal trophic transfer in response to increasing metal contamination may be achieved by a change in metal assimilation efficiency and the ingestion activity of the copepod, but not by changes in metal turnover rates from the animals.

Animals↗

PubMed2000

Determination of interactions between human thrombopoietin and its receptor MPL by yeast two-hybrid system and affinity biosensor.

The binding of human thrombopoietin to the extracellular domain of its receptor MPL prompts a cascade transduction of intracellular signals, leading to the development of megakaryocyte precursors and the production of circulating platelets. We have used a yeast two-hybrid system to reveal, via in vivo interactions between different deletion constructs of MPL and thrombopoietin, that the extracellular subunit 1 of MPL is the ligand binding site and the N-terminal domain of thrombopoietin alone is sufficient for the binding. The extracellular portion of MPL was heterologously expressed in E. coli and its specific affinity with thrombopoietin was visualized in vitro by resonance mirror biosensor technique.

Binding Sites↗

PubMed2000

Cytotoxicity assessment of Ma-huang (Ephedra) under different conditions of preparation.

Ma-huang is a traditional Chinese medicinal herb derived from EPHEDRA: sinica Stapf and other EPHEDRA: species, used to treat asthma, nose and lung congestion, and fever with anhidrosis. It contains 0.5-2.5% by weight of total alkaloids, of which ephedrine accounts for 30 to 90%. Recently, large amounts of ma-huang were used as a source of ephedrine in many dietary supplements formulated for weight reduction, because ephedrine has been found effective in inducing weight loss in diet-restricted obese patients. However, indiscriminate consumption of ma-huang-containing products has resulted in many cases of poisoning, some of which were fatal. The objective of this study is to investigate the relative toxicity of ma-huang extracted under different conditions. The toxicities of various extracts were assayed using MTT colorimetry on a battery of cell lines, while ephedrine alkaloids were analyzed with HPLC. The results are summarized as follows. (1) The cytotoxicity of all ma-huang extracts could not be totally accounted for by their ephedrine contents, suggesting the presence of other toxins in the extracts. (2) Grinding was a significant condition enhancing the toxicity of the extracts. (3) The relatively high sensitivity of the Neuro-2a cell line to the toxicity of ma-huang extracts suggests that the toxic principles were acting on neuronal cells. (4) One condition to produce a ma-huang extract with high ephedrine-to-toxins ratio would be to boil the whole herb for two h.

Cell Survival↗

PubMed1996

Antimutagenicity of ellagic acid against aflatoxin B1 in the Salmonella microsuspension assay.

Ellagic acid (EA) is a phenolic compound with antimutagenic and anticarcinogenic properties. It occurs naturally in some foods such as strawberries, raspberries, grapes, black currants and walnuts. In the present study, we used the Salmonella microsuspension assay to examine the antimutagenicity of EA against the potent mutagen aflatoxin B1 (AFB1) using tester strains TA98 and TA100. Further, we used a two-stage incubation procedure that incorporates washing the bacterial cells free of the incubation mixture after the first incubation to investigate EA and AFB1 interaction. Three different concentrations of AFB1 (2.5, 5 and 10 ng/tube) were tested against five different concentrations of EA for TA98 and TA100. EA significantly inhibited mutagenicity of all doses of AFB1 in both tester strains with the addition of S9. EA alone was not mutagenic at the concentrations tested. The greatest inhibitory effect of EA on AFB1 mutagenicity occurred when EA and AFB1 were incubated together. Lower inhibition was apparent when the cells were first incubated with EA followed by a second incubation with AFB1, and also when the cells were first incubated with AFB1 followed by a second incubation with EA alone. The results of the sequential incubation studies support the hypothesis that one mechanism of inhibition could involve the formation of a chemical complex between EA and AFB1.

Aflatoxin B1↗

PubMed1996

Purification and characterization of the esterases involved in aflatoxin biosynthesis in Aspergillus parasiticus.

The esterases from the cell-free extracts (CFEs) of Aspergillus parasiticus ATCC15517, an aflatoxin-producing strain, catalyzing the hydrolytic conversion of versiconal hemiacetal acetate (VHA) to versiconal was biochemically studied. The specific activity of the enzymes increased 2.5-fold during incubation of mycelia through 40-55 h. No metal ions were required for enzyme stability, but EDTA at 1 mM and dithiothreitol at 0.5-5 mM increased its stability. Three peaks of VHA esterase activity were resolved when the proteins in the CFEs prepared from the mycelia of different ages were separated by anion-exchange column chromatography, suggesting that at least three VHA esterases were present in the eluate of this purification step. One of these esterases extracted from the mycelia of a 55-h culture was partially purified in five steps by means of preparative chromatography and fast protein liquid chromatography. The partially purified enzyme when reacted with [14C]diisopropylfluorophosphate followed by sodium dodecyl sulfate - polyacrylamide gel electrophoresis gave a single radiolabelled band, which corresponded to a protein of 32 kDa. The molecular mass of the partially purified VHA esterase determined with gel filtration was around 60 kDa. The results suggested that the enzyme consists of two isomeric subunits.

Aflatoxins↗

PubMed1995

Comparison of multi-media transport and transformation models: regional fugacity model vs. CalTOX.

Two multimedia environmental transport and transformation computer models are summarized and compared. The regional fugacity model published by Mackay and Paterson (1991), termed Fug3ONT, is a four compartment steady-state model designed to simulate the relative distribution of nonionic organic chemicals in a multimedia system. CalTOX is a seven compartment multimedia total exposure model for hazardous waste sites. Both models are based on the principles of fugacity. CalTOX, however, separates the soil into three layers (surface, root, and vadose) and uses a different approach to estimate the diffusive mass transfer rate in soil. These differences result in lower estimates of the steady-state contaminant concentrations of six environmentally relevant chemicals in the root soil of CalTOX as compared to the bulk soil of Fug3ONT. The difference is greatest for compounds with low mobility in soil such as 2,3,7,8-Tetrachlorodibenzo-p-dioxin and Benzo(a)pyrene where estimates from CalTOX and Fug3ONT differ by more than 3 orders of magnitude. Otherwise, the models provide similar estimates for the distribution of the six chemicals among the air, water, sediment and surface soil.

Air Pollutants↗

PubMed1995

Airborne mutagens produced by frying beef, pork and a soy-based food.

Airborne cooking by-products from frying beef (hamburgers), pork (bacon strips) and soybean-based food (tempeh burgers) were collected, extracted, tested for mutagenicity and chemically analysed. The fumes generated by frying pork and beef were mutagenic, with 4900 and 1300 revertants/g of food cooked, respectively. No mutagenicity was detected in fumes from frying tempeh burgers. Bacon fried to a well-done but non-charred state was eight times more mutagenic in a microsuspension Ames/Salmonella test (TA98 with S-9) than hamburgers and about 350 times more mutagenic than tempeh burgers. Among food samples cooked to a well-done, non-charred state, bacon strips had almost 15-fold more mass (109.5 ng/g) than that of the beef, whereas no heterocyclic amine (HCA) was detected in the fried tempeh burgers. 2-Amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) was the most abundant HCA, followed by 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline (MeIQx) and 2-amino-3,4,8-trimethylimidazo[4,5-f]quinoxaline (DiMeIQx). No 2-amino-9H-pyrido[2,3-b]indole (A alpha C) was detected in the food samples fried at about 200 degrees C, although it was present in the collected airborne products. The total amounts of HCAs in the smoke condensates were 3 ng/g from fried bacon, 0.37 ng/g from fried beef and 0.177 ng/g from fried soy-based food. This study indicates that cooks are potentially exposed to relatively high levels of airborne mutagens and carcinogens and that long-term sampling inside restaurants and kitchens may be warranted in order to assess the potential risk of prolonged exposure.

Air Pollutants↗

PubMed1992

Quantitative integration of the Salmonella microsuspension assay with supercritical fluid extraction of model airborne vapor-phase mutagens.

Vapor-phase mutagens are potentially a major class of toxic contaminants in ambient and indoor air. These compounds are not routinely analyzed due to a lack of an established integrated methodology to quantitatively trap, extract and test the compounds in a bioassay. In a previous report, we emphasized the trapping of volatile and semi-volatile mutagens and the extraction of these compounds using supercritical carbon dioxide (CO2). In the present study, we discuss the use of a bioassay for the quantitation of the model mutagens, ethylene dibromide(EDB) and 4-nitrobiphenyl (4-NB), trapped from an airstream. The compounds EDB and 4-NB were released into a controlled airstream, trapped on XAD-4 adsorbent, and were extracted using supercritical CO2. The extract was tested in a microsuspension modification of the Ames Salmonella/microsome test adapted for volatile compounds. Linear dose-response relationships were obtained for supercritical CO2-extracted EDB using tester strain TA100 (+/- S9) and for 4-NB using tester strains TA98 and TA100 (-S9). Standard dose-response curves with known amounts of the compounds were also determined for comparison with measured amounts of the model compounds collected in an airstream. The gas chromatographic (GC)- and bioassay-determined quantities of EDB and 4-NB were highly correlated, accurate and precise. For example, bioassay-determined EDB concentrations were within 10% of the GC-determined concentrations. Our results demonstrate that the integrated methodology for vapor-phase mutagens developed in this study would be useful for quantitative analysis of these and related airborne vapor-phase mutagenic compounds.

Biphenyl Compounds↗

PubMed1992

Aflatoxin toxicity in cultured human epidermal cells: stimulation by 2,3,7,8-tetrachlorodibenzo-p-dioxin.

Aflatoxin B1 (AFB1) at 1 microgram/ml was markedly toxic to human epidermal cells grown in the Rheinwald-Green 3T3 feeder layer system. At 0.1 microgram/ml, the toxicity was barely evident, as assessed by colony expansion during a 2 week exposure, but it was dramatically stimulated by 5 nM 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), which was non-toxic alone. Neither AFB1-dihydrodiol nor AFB2 were toxic in the presence or absence of TCDD, indicating that metabolism to the 8,9-epoxide was responsible for the AFB1 toxicity. Stimulation of AFB1 epoxidation by TCDD was also indicated by the > 20-fold increase in DNA adduct formation in cultures exposed to [14C]AFB1 and TCDD for 4 days as compared to AFB1 alone. Analysis of free metabolites in culture medium by reverse-phase HPLC revealed that confluent epidermal cultures metabolized AFB1 to AFM1, AFB2a and aflatoxicol. In the presence of TCDD, the levels of AFM1 were higher (14 versus 3% of dose) as were those of AFB2a (3 versus 0.5% of dose), while aflatoxicol levels were lower (0.8 versus 2% of dose). In the absence of irradiated 3T3, the toxicities of AFB1, AFB2, AFM1 and aflatoxicol to cells in serum-free medium (0.15 mM Ca2+) were similar to those in the feeder layer system. Although this moderately low calcium concentration appeared quite satisfactory for observing toxicity, the response was attenuated at a lower calcium concentration (0.09 mM Ca2+).

Aflatoxin B1↗

PubMed1991

Determination of volatile and semivolatile mutagens in air using solid absorbents and supercritical fluid extraction.

Volatile toxicants may be present in emissions from mobile and stationary sources as well as in ambient air. Methods for collecting and concentrating volatiles from air samples have been developed. Solid-phase adsorbents were compared in their trapping efficiencies for dichloromethane (DCM), ethylene dibromide (EDB), 4-nitroblphenyl (4-NB), 2-nitrofluorene (2-NF), and fluoranthene (FI). Charcoal and Carbosieve were the most efficient media for retaining DCM, while XAD-4 was the best adsorbent for EDB and the aromatic compounds. Extraction of direct spikes of compounds from adsorbents using supercritical carbon dioxide resulted in greater than 90% recovery of EDB and 60-92% recovery of the aromatics. Integration of trapping and desorption methods with the Salmonella microsuspension bioassay was demonstrated with EDB and 4-NB recoveries from air; chemical analysis and bioassay gave comparable results (within 10%).

Adsorption↗

PubMed1991

Benzene-induced micronuclei formation in mouse fetal liver blood, peripheral blood, and maternal bone marrow cells.

The transplacental cytogenetic effects of benzene were studied by using the micronucleus test of polychromatic erythrocytes (PCE) found in both fetal liver and fetal peripheral blood, and were compared with PCE from maternal bone marrow. Timed-pregnant mice received single intraperitoneal doses of benzene (0, 109, 219, 437, or 874 mg/kg bw) on the 14th day of gestation and were sacrificed 21 hr after injection. Benzene elicited a significant increase (P less than 0.01) in the frequency of micronucleated polychromatic erythrocytes (MNPCE) in fetal liver blood cells (0.55 to 1.36%, control 0.18%) at doses of 219 to 874 mg/kg, and in fetal peripheral blood cells (0.49 to 0.58%, control 0.25%) and maternal bone marrow cells (0.53 to 0.70%, control 0.10%) at doses of 437 and 874 mg/kg. The data demonstrate that benzene is a moderate transplacental clastogenic agent, and that the mouse transplacental micronucleus test using fetal liver blood cells is a potentially more sensitive indicator of the genotoxicity of benzene than either fetal peripheral blood or maternal bone marrow cells.

Animals↗

PubMed1991

Bisfuranoid mycotoxins: their genotoxicity and carcinogenicity.

Based on the mode of action of AFB1 and the activities of its biologically active intermediates, one may conclude that: 1. The mode of toxic action of the bisfuranoid mycotoxin is through epoxidation of the vinyl ether double bond of their dihydrobisfuran functionality. 2. The DNA and plasma albumin adducts formed in vivo may be useful in the molecular dosimetry of these environmental carcinogens. 3. There appears to be a linear correlation between the steady state levels of AFB1-FAPy-DNA adducts and the carcinogenicity of AFB1. Elucidation of the molecular basis of this correlation may shed light on the mechanism of AFB1-induced carcinogenesis. 4. Consistent appearance of AFB1-DNA adducts in the livers of liver cancer patients tested is supportive of the IARC conclusion that AFB1 is a human carcinogen involved in human primary liver cancer.

Aflatoxin B1↗

PubMed1991

Measuring personal exposure to airborne mutagens and nicotine in environmental tobacco smoke.

The exposure of individuals to environmental tobacco smoke (ETS) is of increasing public health concern because epidemiological studies have associated passive smoking with increased risk of a variety of adverse health effects among non-smokers including lung cancer. As a way to measure individual exposure to the mutagenic compounds in the complex mixture of ETS, we used a sensitive Salmonella/microsome micro pre-incubation (microsuspension) assay to detect mutagenicity of particulate matter collected on filters from low volume (1.7 1/min flow rate) personal sampling pumps. Airborne nicotine was collected concurrently as a marker for ETS exposure. In pilot-field studies, individual exposure to ETS was measured in two separate indoor environments in which smokers were present: a gambling casino and a bingo parlor. Total suspended particulate matter (TSP) was collected on filters worn near the breathing zone of non-smoking individuals. Sampling times ranged from 40 min to 6 h. All extracts of filters had detectable levels of mutagenic activity (TA98, +S9) resulting in airborne mutagenic activity concentrations of 500-5000 rev/m3. The mutagenic activity of the filters from the casino and bingo parlors was significantly correlated with total particulate matter per filters (n = 12; Rho = 0.85, p less than 0.01) and with airborne nicotine per filter (n = 12; Rho = 0.95, p less than 0.01). The microsuspension assay was sufficiently sensitive to detect the mutagens associated with extracts of particulate matter from low volume samples (0.2-0.6 m3) in these indoor environments over a relatively short sampling time, and could be useful in studies of personal exposure to the mutagens in environmental tobacco smoke. Further, airborne nicotine concentrations were highly correlated with airborne mutagenicity and the mutagenic activity associated with ETS could therefore be estimated by the concentrations of nicotine.

Air Pollution↗

PubMed1991

Micronucleus formation in cultured fetal liver blood cells using mitomycin C.

Mouse fetal-liver blood cells were cultured and used to investigate micronucleus formation after exposure to mitomycin C (MMC). The isolated fetal cells were incubated in a medium supplemented with erythropoietin (EPO), and the frequency of micronuclei formation was detected in polychromatic erythrocytes (PCE). The effects of four variables were investigated: (1) MMC exposure dose, (2) MMC exposure time, (3) incubation time, and (4) EPO concentration. PCE were formed by proliferation and differentiation of the erythroid cells in culture. Micronucleated PCE (MNPCE) were observed in a dose-dependent manner after exposing the cultured cells with up to 1.0 microgram/ml MMC. The optimum time of MMC exposure and post-exposure incubation was 3 h and 48 h, respectively, and the optimum EPO concentration was 0.25 U/ml. Mouse fetal-liver PCE are sensitive primordial cell targets that can be obtained in relatively large numbers from a single pregnant animal. The procedure is relatively simple and potentially useful in detecting mutagens and carcinogens capable of causing chromosomal damage.

Animals↗

PubMed1990

Biological reactive intermediates of bisfuranoid mycotoxins.

Aflatoxin B1↗