Localization and distribution of tissue type and urokinase type plasminogen activators and their inhibitors type 1 and 2 in human and rhesus monkey fetal membranes

Fetal membranes consist of 10 distinct layers including components of amnion, chorion and decidua, the latter being of maternal origin. They form mechanically integrated sheets capable of retaining amniotic fluid and play an essential role in protecting fetal growth and development in the pregnant uterus. The extracellular matrix, substrate for plasminogen activators (PAs), is an important supportive framework of the fetal membranes. :Fetal membranes from women with preterm premature rupture of membranes may differ in their protease activity compared with normal membranes. To identify the presence of PAs and their inhibitors (PAI) and their possible role in the process of fetal membrane rupture, this study in investigated the distribution and localization of both protein and mRNA for tissue (t) and urokinase (u) PA and their inhibitors type 1 (PAI-1) and type 2 (PAI-2) in amniochorion of human and rhesus monkey using conventional and. confocal immunofluorescence microscopy. In situ hybridization analysis showed that the distribution and localization of mRNAs for tPA, uPA, PAI-I and PAI-2 were similar in the fetal membranes of human and rhesus monkey; no obvious species difference was observed. Evidence of tPA mRNA was detected in amniotic epithelium, trophoblast cells and nearly all cells of the decidual layer. Strong expression of uPA mRNA was noted in the decidual cells which increased in intensity as the abscission point was approached. Weak staining in chorion laeve trophoblast was also detected. In situ hybridization experiments showed PAI-1 mRNA to be concentrated mainly in the decidual cells, some of which were interposed into the maternal-facing edge of the chorion laeve. Maximal labelling of the decidua occurred towards the zone of abscission. Weak expression of PAI-1 mRNA nas also noted in some cells of the chorion laeve. The distribution of PAI-2 mRNA in amniochorion was also concentrated in the cells of the decidual layer, maximum expression of the mRNA was in the level of abscission. No detectable amount of mRNAs for tPA, uPA, PAI-1 and PAI-2 was found in the fibroblast, reticular and spongy layers. Distribution of the proteins of tPA, uPA and PAI-1 in the fetal membranes of these two species was consistent with the distribution of their mRNA. Anti-PAI-2 immunofluorescence was found to be strongly concentrated in the amniotic epithelium, but PAI-2 mRNA was negative in this layer, suggesting that the epithelium-associated PAI-2 is not of epithelial origin. These findings suggest that a local fibrinolysis in fetal membranes generated by precisely balanced expression of PAs and their inhibitors via paracrine or autocrine mechanisms may play an essential role in fetal membrane development, maturation and in membrane rupture. Following an analysis of the distribution and synthesis of activators and inhibitors it was found that they may play a role in abscission during the third stage of labour. (C) 1998 W. B. Saunders Company Ltd.

Effects of aniracetam on extracellular levels of transmitter amino acids in the hippocampus of the conscious gerbils: an intracranial microdialysis study

The effects of aniracetam on extracellular amino acid levels in the hippocampus of conscious gerbils, with or without transient cerebral ischemia/reperfusion, were measured by microdialysis and reverse phase-high performance liquid chromatography. Increased extracellular levels of aspartate and glutamate that were observed in the hippocampus of conscious gerbils during transient global forebrain ischemia were reversed by aniracetam. In contrast, the level of extracellular gamma-aminobutyric acid was increased, while taurine was maintained at a higher level than other amino acids by administration of aniracetam (100 mg/kg, p.o.) 60 min before ischemia. Further, in contrast to ischemic animals, administration of aniracetam (100 mg/kg, p.o.) enhanced the release of glutamate and aspartate in the normal gerbil hippocampus. The results suggest that these effects might be due to a partial calcium agonist activity of aniracetam, and that the effects of aniracetam on amino acid levels might be a mechanism of protection against delayed neuronal death in the ischemic hippocampus, thereby improving memory dysfunction induced by ischemia/reperfusion. (C) 2003 Elsevier Science Ireland Ltd. All rights reserved.

DE-71-induced apoptosis involving intracellular calcium and the Bax-mitochondria-caspase protease pathway in human neuroblastoma cells In vitro

Polybrominated diphenyl ethers (PBDEs) are used extensively as flame-retardants and are ubiquitous in the environment and in wildlife and human tissue. Recent studies have shown that PBDEs induce neurotoxic effects in vivo and apoptosis in vitro. However, the signaling mechanisms responsible for these events are still unclear. In this study, we investigated the action of a commercial mixture of PBDEs (pentabrominated diphenyl ether, DE-71) on a human neuroblastoma cell line, SK-N-SH. A cell viability test showed a dose-dependent increase in lactate dehydrogenase leakage and 3-(4,5-dimethylthia-zol-2-yl)-2,5-diphenyl-tetrazolium bromide reduction. Cell apoptosis was observed through morphological examination, and DNA degradation in the cell cycle and cell apoptosis were demonstrated using flow cytometry and DNA laddering. The formation of reactive oxygen species was not observed, but DE-71 was found to significantly induce caspase-3, -8, and -9 activity, which suggests that apoptosis is not induced by oxidative stress but via a caspase-dependent pathway. We further investigated the intracellular calcium ([Ca2+](i)) levels using flow cytometry and observed an increase in the intracellular Ca2+ concentration with a time-dependent trend. We also found that the N-methyl d-aspartate (NMDA) receptor antagonist MK801 (3 mu M) significantly reduced DE-71-induced cell apoptosis. The results of a Western blotting test demonstrated that DE-71 treatment increases the level of Bax translocation to the mitochondria in a dose-dependent fashion and stimulates the release of cytochrome c (Cyt c) from the mitochondria into the cytoplasm. Overall, our results indicate that DE-71 induces the apoptosis of ([Ca2+](i)) in SK-N-SH cells via Bax insertion, Cyt c release in the mitochondria, and the caspase activation pathway.

PCBs and OCPs in human milk and selected foods from Luqiao and Pingqiao in Zhejiang, China

This study was conducted to measure the levels of 23 PCB congeners and 6 organochlorine pesticides (OCPs) in human milk and three food types collected from Luqiao and Pingqiao in Zhejiang Province, China. An effort was also made to explore the potential health risk for the mothers and breast-fed infants living in these two localities. Luqiao was selected as the sampling site because it is the largest place for the disassembly of obsolete transformers and electrical waste in China. Pingqiao, located 100 kin NW of Luqiao, is not known to be a place for any electronic or electrical waste and hence was chosen as the control site. Both localities are important agricultural places in the province. The organochlorines were measured in the samples using the GC-PECD technique. Micro-EROD bioassay method was also used as a complement of the chemical analysis to estimate the TEQ levels of dioxin-like PCBs in human milk. The data showed that the human milk, rice, hen egg, and fish samples from Luqiao were more heavily contaminated with PCBs than those from Pingqiao, suggesting that the mothers and their breast-fed infants in Luqiao tended to receive greater exposure to PCBs than those living in Pingqiao. The OCP levels in the two localities were found comparable, suggesting that the major source of contamination with these pesticides was from their agricultural uses. Significant correlation (R-2 = 0.87, P < 0.001) of PCB TEQs was found between the bioassay and chemical analysis method, suggesting that micro-EROD is an effective method for comprehensive determination of TEQ levels in human milk. Comparison with literature data showed that the PCB levels in milk samples from Luqiao were significantly higher than those from localities in other Chinese provinces and comparable to those in developed or industrialized countries. (c) 2007 Published by Elsevier B.V.

An improved method for the extraction of low molecular weight organic acids in variable charge soils

Due to specific adsorption to variable charge soils, low molecular weight organic acids (LMWOAs) have not been sufficiently extracted, even if common extractants, such as water and 0.1 M sodium hydroxide (NaOH), were employed. In this work, the method for extracting LMWOAs in soils with 0.1 M NaOH was improved for variable charge soils; e.g. 1.0 M potassium fluoride (KF) with pH 4.0 was applied as an extractant jointed with 0.1 M NaOH based on its stronger ability to change the electrochemical properties of variable charge soils by specific adsorption. With the proposed method, the recoveries of oxalic, tartaric, malic, citric and fumaric acids were increased from 83 4, 93 1, 22 2, 63 +/- 5 and 84 +/- 3% to 98 +/- 2, 100 +/- 2, 85 +/- 2, 90 +/- 2 and 89 +/- 2%, respectively, compared with NaOH alone. Simultaneously, the LMWOAs in Agri-Udic Ferrosol with field moisture were measured with a satisfactory result.

Optimization of RP-HPLC analysis of low molecular weight organic acids in soil

RP-HPLC analysis for low molecular weight organic acids in soil solution has been optimized. An Atlantis (TM) C-18 column was used for the analyses. An optimal determination for eleven organic acids in soil solution was found at room temperature (25 degrees C) and 220 nm detection wavelength, with a mobile phase of 10 mM KH2PO4 -CH3OH (955, pH 2.7), a flow rate of 0.8 mL/min and 10 mu L sample size. The detection limits ranged 3.2-619 ng/mL, the coefficients of variation ranged 1.3-4.6%, and the recoveries ranged 95.6-106.3% for soil solution with standard addition on the optimal conditions proposed.

Enhanced resistance to Aeromonas hydrophila infection and enhanced phagocytic activities in human lactoferrin-transgenic grass carp (Ctenopharyngodon idellus)

Human lactoferrin (hLF) is an iron-binding protein with antimicrobial and immunomodulatory activities. hLF cDNA was transferred into grass carp via electroporated sperm. The production of transgenic fish was as high as 55% tinder the best parameters. 2(11) pulses and 20-min incubation. The expression of the transgene was demonstrated by the detection of hLF mRNA by RT-PCR. We also investigated the response of G(0) transgenic grass carp to Aeromonas hydrophila infection. Serum lysozyme activities (P>0.05) and phagocytic activities of kidney cells (P<0.05) were measured in transgenic individuals. The transgenic fish not only cleared A. hydrophila significantly faster than the control carp (P<0.05), but also showed enhanced phagocytic activities. The result shows that hLF has immunomodulatory activities in hLF-transgenic grass carp. The transgenic grass carp exhibited enhanced immunity to A. hydrophila infection. These results reveal that the mechanisms of disease resistance are different between hLF-transgenic plants and hLF-transgenic grass carp. (C) 2004 Elsevier B.V. All rights reserved.

Dioxin-like components in human breast milk collected from Hong Kong and Guangzhou

The H4IIE rat hepatoma cell line was employed as a cell model to screen 7-ethoxyresorufin O-deethylase (EROD)-TCDD equivalents (EROD-TEQ) of human breast milk samples collected from Hong Kong and Guangzhou, China. The screening methods employed a 96-well plate spectrofluorometer-EROD assay. For cell-line validation, our results demonstrated a dose-dependent increase in the Ah receptor-mediated response (i.e., CYP1A1 mRNA and EROD) of the cells upon exposure to a number of known Ah receptor agonists, including 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), 2,3,7,8-tetrachlorodibenzothiophene, benzo[a]pyrene, and beta-naphthaflavone. TCDD induced CYP1A1 mRNA and EROD was in a close positive correlation (r = 0.98). For the screening of dioxin-like compounds, breast milk samples collected during lactation weeks 3-5 were used. One hundred (from Hong Kong) and 48 (from Guangzhou) breast milk samples were assayed, of which 65% and 68% of the samples, respectively, showed detectable dioxin-like activities using the H4IIE cell EROD screening method. For sixty-five samples from Hong Kong the mean EROD-TEQ values ranged from 58.1 to 96.5 pg/g of milk fat for those aged 21-36 years while 32 samples from Guangzhou had mean values of 98.8-202.1 pg/g of milk fat. In comparisons of the EROD-TEQ values for different age groups from both cities, there were no significant differences (P < 0.05). However, the mean and median EROD-TEQ values of the Guangzhou population were in general higher than those of the Hong Kong population. The results of the present study indicate that it is feasible to use the H4IIE cell-line as a model for screening dioxin-like compounds in human breast milk. In addition, the method is rapid and cost-effective, particularly for a routine and high-throughput sample screening analysis, compared to the costly and time-intensive chemical analytical techniques. (C) 2003 Elsevier Inc. All rights reserved.

High-efficiency transfer and expression of AdCMV-p53 in human cervix adenocarcinoma cells induced by subclinical-dose carbon beam radiation

Purpose The aim of this study is to evaluate the eVect of carbon-beam irradiation on adenovirus-mediated p53 transfer in human cervix adenocarcinoma.Materials and methods The HeLa cells pre-exposed to carbon-beam or -ray, were infected with replication-deficient adenovirus recombinant vectors, containing human wild-type p53 (AdCMV-p53) and green Xuorescent protein (GFP) (AdCMV–GFP), respectively. The GFP transfer and p53 expression were detected by Xow cytometric analysis.Results The GFP transfer frequency in C-beam with AdCMV-GFP groups was 38–50% more than that inγ-ray with AdCMV–GFP groups. The percentage of p53 positive cells in the C-beam with AdCMV–p53 groups was 34–55.6% more than that in γ-ray with AdCMV-p53 groups (p < 0.05), suggesting that subclinical-dose C-beam irradiation could signiWcantly promote exogenous p53 transfer and p53 expression, and extend the duration of p53 expression in the HeLa cells. The expression of p21 increased with p53 expression in HeLa cells. The survival fractions for the 0.5–1.0 Gy C-beam with AdCMV-p53 groups were 38–43% less than those for the isodose γ-ray with AdCMV-p53 groups, and 31–40% less than those for the C-beam only groups (p <0.05).Conclusions The subclinical-dose C-beam irradiation could signiWcantly promote the transfer and expression of exogenous p53, extend the duration of p53 expression, and enhance the suppression of p53 on cervix adenocarcinoma cells.

Characteristics and sources of formic；acetic and oxalic acids in PM2.5 and PM10 aerosols in Beijing；China

IEECAS SKLLQG

Micronuclei induction in human lymphocytes induced by carbon ions exposion along the penetrate depth of ions in water

Here we used cytokinesis-block micronucleus assay to measure the biological response along the penetrate depth of ions in water in human lymphocytes exposed to 100 MeV/u incident carbon ions in vitro. Polyethylene shielding was used to change the penetration depth of ions in water. A quantitative biological response curve was generated for micronuclei induction. The results showed a marked increase with the penetrate depth of ions in water in the micronuclei formation, which was consistent with a linearenergy- transfer dependent increase in biological effectiveness. The dose–response relationship for MN information was different at different penetrate depth of ions in water, at the 6 and 11.2 mm penetrate depth of ions in water, the dose–response relationships for the micronucleus frequencies induced by carbon ions irradiation were linear; while it was power function at 17.1 mm penetrate depth.

Prematurely condensed chromosome fragments in human lymphocytes induced by high doses of high-linear-energy-transfer irradiation

This study provides a useful biodosimetry protocol for radiation accidents that involve high doses of heavy particle radiation. Human peripheral blood lymphocytes (PBLs) were irradiated in vitro with high doses (5–50 Gy) of charged heavy-ion particles (carbon ions, at an effective linear-energy-transfer (LET) of 34.6 keV/ m), and were then stimulated to obtain dividing cells. PBLs were treated with 100nMcalyculin A to force chromosomes to condense prematurely, and chromosome spreads were obtained and stained with Giemsa. The G2 prematurely condensed chromosome (G2-PCC) index and the number of G2-PCC including fragments (G2-PCC-Fs) per cell for each radiation dose point were scored. Dose-effect relationships were obtained by plotting the G2-PCC indices or G2-PCC-Fs numbers against radiation doses. The G2-PCC index was greater than 5% up to doses of 15 Gy; even after a 30Gy radiation dose, the index was 1 to 2%. At doses higher than 30 Gy, however, the G2-PCC indices were close to zero. The number of G2-PCC-Fs increased steeply for radiation doses up to 30 Gy at a rate of 1.07 Gy−1. At doses higher than 30 Gy, the numbers of G2-PCC-Fs could not be accurately indexed because of the limited numbers of cells for analysis. Therefore, the number of G2-PCC-Fs could be used to estimate radiation doses up to 30 Gy. In addition, a G2-PCC index close to zero could be used as an indicator for radiation doses greater than 40 Gy.