Use of sequential ASE extraction to evaluate the bioavailability of DDT and its metabolites to wheat roots in soils with various organic carbon contents.

An accelerated solvent extraction (ASE) procedure using water, n-hexane and a mixture of n-hexane and acetone as solvents in sequence was developed and tested to evaluate the bioavailability of DDT and its metabolites including p,p'-DDT, o,p'-DDT, p,p'-DDE, and p,p'-DDD (SigmaDDTs) to wheat uptake from soils characterized by varied organic carbon contents. Results indicated that the extractability of SigmaDDTs with water was enhanced considerably in the presence of water soluble organic carbon (WSOC), while the amount of SigmaDDTs extracted with n-hexane was negatively correlated to the content of water insoluble organic carbon (WIOC). The interaction between SigmaDDTs and WIOC also reduced the bioavailability of the pesticides to wheat roots during uptake. There was a good positive correlation between the amount of SigmaDDTs extracted by n-hexane and the amount of SigmaDDTs accumulated in wheat roots, suggesting some potential for the use of the n-hexane ASE-extracted fraction as an indicator of SigmaDDTs' bioavailability to plant uptake. As such, the three sequentially extracted fractions may be viewed as representing the mobile, bioavailable, and fixed pools of SigmaDDTs in the soil.

Opposing changes in N-acetylglucosaminyltransferase-V and -III during the cell cycle and all-trans retinoic acid treatment of hepatocarcinoma cell line.

The changes in N-acetylglucosaminyltransferase-V and -III (GnT-V, GnT-III) during the cell-cycle of synchronized 7721 human hepatocarcinoma cell line were investigated. Using an HPLC method to assay GnT and flow cytometry (FCM) for cell cycle analysis, it was found that GnT-V showed the highest activity, but GnT-III reached the lowest activity when G(2)/M cells were most abundant. In contrast, GnT-V declined to the minimum while GnT-III elevated to maximum when G(0)/G(1) cells were most predominant. The opposing changes were more obvious when the activities of GnT-V and GnT-III were expressed as relative activities (activity of GnT-V or GnT-III/the sum of activities of GnT-V plus GnT-IV plus GnT-III). These opposing changes of GnT-V and GnT-III during the cell cycle might result from the different regulatory mechanisms of GnT-V and GnT-III expression in the cell cycle. The alterations in the structures of cell surface N-glycans were compatible with the changes of the activities of GnTs. The results from immunocytochemistry and Northern blot showed that the protein and mRNA contents of GnT-V were not significantly changed during the cell cycle. The activity of a cell cycle regulating protein kinase, p34(cdc2) kinase, correlated to the activity of GnT-V. These findings suggested that the change of GnT-V activity in cell cycle was not the consequence of the alteration of gene transcription or enzyme protein synthesis, but might be caused by the post-translational regulation. The decrease in GnT-V and the corresponding increase in GnT-III activities were also found after the cells were treated with all-trans retinoic acid (ATRA), and the mechanism of this might be different from that in the cell cycle.

Regulation of N-acetylglucosaminyltransferase V by protein kinases.

When 7721 human hepatocarcinoma cells were treated with 100 nM phorbol-12-myristate-13-acetate (PMA), the activity of N-acetylglucosaminyltransferase V(GnT-V) in the cells varied in accordance with the activity of membranous protein kinase C (PKC), but not with that of cytosolic PKC. Quercetin, a non-specific inhibitor of Ser/Thr protein kinase, and D-sphingosine and staurosporine, two specific inhibitors of PKC, blocked the activation of membranous PKC and GnT-V by PMA. Among the three inhibitors, quercetin was least effective. The inhibitory rates of quercetin and staurosporine toward membranous PKC and GnTV were proportional to the concentrations of the two inhibitors. The activities of GnTV and membranous protein kinase A (PKA) were also induced in parallel by dibutyryl cAMP (db-cAMP) and this induction was blocked by a specific PKA inhibitor. When cell free preparations of 7721 cells and human kidney were treated with alkaline phosphatase (ALP) to remove the phosphate groups, the GnTV activities were decreased. These results suggest that GnTV may be activated by membranous PKC or PKA, indirectly or directly, via phosphorylation of Ser/Thr residues.

Effect of retinoic acid on the structure of N-glycans on the surface of human hepatocarcinoma cells and its enzymatic mechanism.

In order to study the effect of retinoic acid on the structure of N-glycans on the cell surface, the N-glycans of glycoproteins on the surface of 7721 human hepatocarcinoma cells were labelled with [3H] mannose, added to the culture medium. The 3H-labelled N-glycans were prepared from cell-surface glycoproteins, desialylated, and subjected to sequential chromatography on concanavalin A and Datura stranonium agglutinin affinity columns to separate the glycans into four fractions of different type and different antennary number. It was found that the percentage of C2C2 biantennary complex type N-glycans was increased, but the high-mannose type as well as the tri- and tetraantennary complex types, especially that with a C2.6 branched structure, were decreased after the cells had been treated with retinoic acid for 3-5 days. Using a Lens culinaris agglutinin affinity column, it was discovered that the core fucose in the biantennary glycan was also decreased. The enzymatic mechanisms of the above changes were revealed in further study to involve the decrease of N-acetylglucosaminyl-transferase V and core alpha-1,6-fucosyltransferase by retinoic acid.