Treatment of trichloroethylene (TCE) in a membrane biofilter

This article reports on the biodegradation of trichloroethylene (TCE) in a hollow-fiber membrane biofilter. Air contaminated with TCE was passed through microporous hollow fibers while an oxygen-free nutrient solution was recirculated through the shell side of the membrane module. The biomass was attached to the outside surface of the microporous hollow fibers by initially supplying toluene in the gas phase that flows through the fibers. While studies on TCE biodegradation were conducted, there was no toluene present in the gas phase. At 20-ppmv inlet concentration of TCE and 36-s gas-phase residence time, based on total internal volume of the hollow fibers, 30% removal efficiency of TCE was attained. At higher air flow rates or lower gas-phase residence times, lower removal efficiencies were observed. During TCE degradation, the pH of the liquid phase on the shell side of the membrane module decreased due to release of chloride ions. A mathematical model was developed to describe the synchronous aerobic/anaerobic biodegradation of TCE. (c) 1996 John Wiley & Sons, Inc.     

Identification and characterization of a mitochondrial targeting signal in rat cytochrome P450 2E1 (CYP2E1)

Cytochrome P450 2E1 (CYP2E1) lacking the hydrophobic NH(2)-terminal hydrophobic transmembrane domain is specifically targeted to mitochondria, where it is processed to a soluble and catalytically active form (Delta2E1) with a mass of about 40 kDa. Small amounts of Delta2E1 were also observed in mitochondria isolated from rat liver, indicating that this form of CYP2E1 is also present in vivo. In the present study the mitochondrial targeting signal was identified and characterized by the use of several NH(2)-terminally truncated and mutated forms of CYP2E1 that were expressed in the mouse H2.35 hepatoma cell line. Two potential mitochondrial targeting sequences were identified in the NH(2) terminus of CYP2E1. Deletion of the first potential mitochondrial targeting sequence located between amino acids 50 and 65, as in Delta(2-64)2E1, still resulted in mitochondrial targeting and processing, but when, in addition to the first, the second potential mitochondrial targeting sequence located between amino acids 74 and 95 was also deleted, as in Delta(2-95)2E1, the mitochondrial targeting was abolished. Mutation of the four positively charged Arg and Lys residues present in this sequence to neutral Ala residues resulted in the abrogation of mitochondrial targeting. Deletion of a hydrophobic stretch of amino acids between residues 76 and 83 also abolished mitochondrial targeting and import. Once imported in the mitochondria, these constructs were further processed to the mature protein Delta2E1. It is concluded that mitochondrial targeting of CYP2E1 is mediated through a sequence located between residues 74 and 95 and that positively charged residues as well as a hydrophobic stretch present in the beginning of this sequence are essential for this process.     

Linkage relationships of the apolipoprotein C1 gene and a cytochrome P450 gene (CYP2A) to myotonic dystrophy

Summary We have studied the genetic linkage of two markers, the apolipoprotein C1 (APOC1) gene and a cytochrome P450 (CYP2A) gene, in relation to the gene for myotonic dystrophy (DM). A peak lod score of 9.29 at 2 cM was observed for APOC1-DM, with a lod score of 8.55 at 4cM for CYP2A-DM. These two markers also show close linkage to each other ( _max = 0.05, Z _max = 9.09). From examination of the genotypes of the recombinant individuals, CYP2A appears to map proximal to DM because in one recombinant individual CYP2A, APOC2 and CKMM had all recombined with DM. Evidence from another CYP2A-DM recombinant individual places CYP2A proximal to APOC2 and CKMM. Localisation of CYP2A on a panel of somatic cell hybrids also suggests that it is proximal to DM and APOC2/C1/E gene cluster.     

Phytoremediation of trichloroethylene and dichlorodiphenyltrichloroethane-polluted water using transgenic Sesbania grandiflora and Arabidopsis thaliana plants harboring rabbit cytochrome p450 2E1

Sesbania grandiflora (L.) pers (Fabaceae) and Arabidopsis thaliana (L.) (Brassicaceae) were genetically engineered to constitutively express the rabbit cytochrome p450 2E1 enzyme aiming at increasing their activity toward trichloroethylene (TCE) and dichlorodiphenyltrichloroethane (DDT) removal Successful generation of Sesbania and Arabidopsis transgenic plants was verified using p450 2E1 specific PCR and confirmed by western blot analysis. Gas chromatography (GC) analysis revealed that small cuttings of Sesbania and third generation (F3) Arabidopsis transgenic plants exposed to TCE and DDT in small hydroponics' vessels accumulated more TCE and DDT compared to plants transformed with the empty vector. Furthermore, both transgenic plants were more effective in breaking down TCE and DDT with a 2-fold increase in TCE metabolism. Two independent Arabidopsis lines showed that DDT was metabolized about 4-fold higher than that detected in non transformed plants. Similarly, S. grandiflora cuttings removed 51 to 90% of the added DDT compared with only 3% removal in controls transformed with the null vector. Notably, stability of rabbit cytochrome p450 2E1 was confirmed using third generation Arabidopsis plants that displayed higher potential for the removal of two important pollutants, TCE and DDT compared with the controls.     

E2F1 regulation of the human myo-inositol 1-phosphate synthase (ISYNA1) gene promoter

Human myo-inositol 1-phosphate synthase (IP synthase; E.C. 5.5.1.4), encoded by ISYNA1, catalyzes the de novo synthesis of inositol 1-phosphate from glucose 6-phosphate. It is a potential target for mood-stabilizing drugs such as lithium and valproate. But, very little is known about the regulation of human IP synthase. Here, we have characterized the minimal promoter of ISYNA1 and show that it is upregulated by E2F1. Upregulation occurs in a dose-dependent fashion and can be suppressed by ectopic expression of Rb. EMSA and antibody supershift analysis identified a functional E2F binding motif at -117. Complex formation at this site was competed by an excess of unlabeled Sp1 oligo consistent with the -117 E2F site overlapping an Sp1 motif. Because the -117 E2F motif is not a high-affinity binding site, we propose that the upregulation of ISYNA1 occurs through the cooperative interaction of several low-affinity E2F binding motifs present in the minimal promoter.     

Genetic polymorphism analysis of cytochrome P4502E1 (CYP2E1) in Chinese Han populations from four different geographic areas of Mainland China

CYP2E1 is one of a superfamily of enzymes that play a central role in activating and detoxifying many xenobiotics and endogenous compounds thought to be involved in the development of several human diseases. Among other factors, individual susceptibility to developing these pathologies relies on genetic polymorphisms, which are related to ethnic differences, since the frequency of mutant genotypes varies in different populations. The aim of this study was to investigate the genetic basis of CYP2E1 polymorphisms in the populations of four different geographical locations of China. Twenty-two different CYP2E1 polymorphisms, including six novel variants in promoter regions and a novel nonsense mutation, were identified. The frequencies of some polymorphisms and genotypes demonstrated significant differences among the four populations. Linkage disequilibrium analysis and tag SNP selection were performed. Haplotypes were analyzed within the selected tag SNPs. Tag SNP selection and haplotype distributions showed differences across the four populations.     

Interaction of CYP11B1 (cytochrome P-45011 beta) with CYP11A1 (cytochrome P-450scc) in COS-1 cells.

The interactions of CYP11B1 (cytochrome P-45011beta), CYP11B2 (cytochrome P-450aldo) and CYP11A1 (cytochrome P-450scc) were investigated by cotransfection of their cDNA into COS-1 cells. The effect of CYP11A1 on CYP11B isozymes was examined by studying the conversion of 11-deoxycorticosterone to corticosterone, 18-hydroxycorticosterone and aldosterone. It was shown that when human or bovine CYP11B1 and CYP11A1 were cotransfected they competed for the reducing equivalents from the limiting source contained in COS-1 cells; this resulted in a decrease of the CYP11B activities without changes in the product formation patterns. The competition of human CYP11A1 with human CYP11B1 and CYP11B2 could be diminished with excess expression of bovine adrenodoxin. However, the coexpression of bovine CYP11B1 and CYP11A1 in the presence of adrenodoxin resulted in a stimulation of 11beta-hydroxylation activity of CYP11B1 and in a decrease of the 18-hydroxycorticosterone and aldosterone formation. These results suggest that the interactions of CYP11A1 with CYP11B1 and CYP11B2 do not have an identical regulatory function in human and in bovine adrenal tissue.     

Augmented oxygen-mediated transcriptional activation of cytochrome P450 (CYP)1A expression and increased susceptibilities to hyperoxic lung injury in transgenic mice carrying the human CYP1A1 or mouse 1A2 promoter in vivo

Background/aims

TEC, a member of the TEC family of non-receptor type protein tyrosine kinases, has recently been suggested to play a role in hepatocyte proliferation and liver regeneration. This study aims to investigate the putative mechanisms of TEC kinase regulation of hepatocyte differentiation, i.e. to explore which signaling pathway TEC is involved in, and how TEC is activated in hepatocyte after hepatectomy and hepatocyte growth factor (HGF) stimulation.

Methods

We performed immunoprecipitation (IP) and immunoblotting (IB) to examine TEC tyrosine phosphorylation after partial hepatectomy in mice and HGF stimulation in WB F-344 hepatic cells. The TEC kinase activity was determined by in vitro kinase assay. Reporter gene assay, antisense oligonucleotide and TEC dominant negative mutant (TEC^KM) were used to examine the possible signaling pathways in which TEC is involved. The cell proliferation rate was evaluated by ³H-TdR incorporation.

Results

TEC phosphorylation and kinase activity were increased in 1 h after hepatectomy or HGF treatment. TEC enhanced the activity of Elk and serum response element (SRE). Inhibition of MEK1 suppressed TEC phosphorylation. Blocking TEC activity dramatically decreased the activation of Erk. Reduced TEC kinase activity also suppressed the proliferation of WB F-344 cells. These results suggest TEC is involved in the Ras-MAPK pathway and acts between MEK1 and Erk.

Conclusions

TEC promotes hepatocyte proliferation and regeneration and is involved in HGF-induced Erk signaling pathway.     

Overexpression of the PtSOS2 gene improves tolerance to salt stress in transgenic poplar plants

In higher plants, the salt overly sensitive (SOS) signalling pathway plays a crucial role in maintaining ion homoeostasis and conferring salt tolerance under salinity condition. Previously, we functionally characterized the conserved SOS pathway in the woody plant Populus trichocarpa. In this study, we demonstrate that overexpression of the constitutively active form of PtSOS2 (PtSOS2TD), one of the key components of this pathway, significantly increased salt tolerance in aspen hybrid clone Shanxin Yang (Populus davidiana × Populus bolleana). Compared to the wild‐type control, transgenic plants constitutively expressing PtSOS2TD exhibited more vigorous growth and produced greater biomass in the presence of high concentrations of NaCl. The improved salt tolerance was associated with a decreased Na⁺ accumulation in the leaves of transgenic plants. Further analyses revealed that plasma membrane Na⁺/H⁺ exchange activity and Na⁺ efflux in transgenic plants were significantly higher than those in the wild‐type plants. Moreover, transgenic plants showed improved capacity in scavenging reactive oxygen species (ROS) generated by salt stress. Taken together, our results suggest that PtSOS2 could serve as an ideal target gene to genetically engineer salt‐tolerant trees.