Generation of 2,3,7,8-TCDD-metabolizing enzyme by modifying rat CYP1A1 through site-directed mutagenesis

Polychlorinated dibenzo-p-dioxins (PCDDs) are known as g environmental contaminants on account of the extreme toxicity. Among these compounds, 2,3,7,8-tetrachlorodibenzo-p-dioxin (2,3,7,8-TetraCDD) is regarded as the most toxic one. The extremely high toxicity of 2,3,7,8-TetraCDD is based on its high affinity for Ah receptor and nearly undetectable metabolism in mammalian body. Based on our previous studies, we assumed that enlarging the space of substrate-binding pocket of rat CYP1A1 might generate the catalytic activity toward 2,3,7,8-TetraCDD. Large-sized amino acid residues located at putative substrate-binding sites of rat CYP1A1 were substituted for alanine by site-directed mutagenesis. Among eight mutants examined, the mutant in the putative F-G loop, F240A, showed metabolic activity toward 2,3,7,8-TetraCDD. HPLC and GC-MS analyses strongly suggested that the metabolite was 8-hydroxy-2,3,7-TriCDD. Ah receptor assay revealed that the affinity of 8-hydroxy-2,3,7-TriCDD for Ah receptor was less than 0.01% of 2,3,7,8-TetraCDD, indicating that the F240A-dependent metabolism resulted in remarkable detoxification of 2,3,7,8-TetraCDD. The novel 2,3,7,8-TetraCDD-metabolizing enzyme could be applicable to bioremediation of contaminated soils with dioxin, elimination of dioxin from foods, and clinical treatment for people who accidentally take dioxin into their systems.     

Herbicide-resistant tobacco plants expressing the fused enzyme between rat cytochrome P4501A1 (CYP1A1) and yeast NADPH-cytochrome P450 oxidoreductase.

Transgenic tobacco (Nicotiana tabacum cv Xanthi) plants expressing a genetically engineered fused enzyme between rat cytochrome P4501A1 (CYP1A1) and yeast NADPH-cytochrome P450 oxidoreductase were produced. The expression plasmid pGFC2 for the fused enzyme was constructed by insertion of the corresponding cDNA into the expression vector pNG01 under the control of the cauliflower mosaic virus 35S promoter and nopaline synthase gene terminator. The fused enzyme cDNA was integrated into tobacco genomes by Agrobacterium infection techniques. In transgenic tobacco plants, the fused enzyme protein was localized primarily in the microsomal fraction. The microsomal monooxygenase activities were approximately 10 times higher toward both 7-ethoxycoumarin and benzo[a]pyrene than in the control plant. The transgenic plants also showed resistance to the herbicide chlortoluron.     

Effects of Acriflavine on the Formation of the Plasma Membrane of Escherichia coli

When cells of acriflavine-sensitive (acrA) and acriflavine-resistant(acrA⁺) Escherichia coli K-12 strains were treated with a ratherhigh concentration (100 µg ml^-1) of acriflavine in mediumthat had been adjusted to pH 8.1, distinct whirlpool-like structuresderived from the plasma membrane appeared not only in the acrAcells but also in the acrA⁺ cells. Chemical analysis was performedto determine the lipid composition of the cells by thin-layerchromatography on silica gel and gas-liquid chromatography.The amount of total fatty acids was significantly higher inthe acrA cells than in the acrA⁺ cells, when cells were culturedin the presence of acriflavine. This difference seems to becaused by the greater accumulation of unsaturated fatty acids(palmitoleic and cis-vaccenic acid) in the acrA mutant cellsthan in the acrA⁺ cells and by the acceleration of this accumulationas a result of the presence of the dye. A comparison of phospholipidcontents between the acrA and acrA⁺ cells cultured under acriflavine-freeconditions showed that the former cells contained more phosphatidylethanolamine(PE) and, in particular, more cardiolipin (CL) than the lattercells. However, the situation was reversed in the case of phosphatidylglycerol(PG). Addition of acriflavine to the medium led to a markedincrease in levels of PE and CL in both acrA and acrA⁺ cellsbut an increase in levels of PG was found only in the acrA⁺cells. (Received October 13, 1992; Accepted May 31, 1993)     

Multiple L1 progenitors in prosimian primates: Phylogenetic evidence from ORF1 sequences

One of the uncertainties regarding the evolution of L1 elements is whether there are numerous progenitor genes. We present phylogenetic evidence from ORF1 sequences of slow loris (Nycticebus coucang) and galago (Galago crassicaudatus) that there were at least two distinct progenitors, active at the same time, in the ancestor of this family of prosimian primates. A maximum parsimony analysis that included representative L1s from human, rabbit, and rodents, along with the prosimian sequences, revealed that one of the galago L1s (Gc11) grouped very strongly with the slow loris sequences. The remaining galago elements formed their own unique and strongly supported clade. An analysis of replacement and silent site changes for each link of the most parsimonious tree indicated that during the descent of the Gc11 sequence approximately two times more synonymous than nonsynonymous substitutions had occurred, implying that the Gc11 founder was functional for some time after the split of galago and slow loris. Strong purifying selection was also evident on the galago branch of the tree. These data indicate that there were two distinct and contemporaneous L1 progenitors in the lorisoid ancestor, evolving under purifying selection, that were retained as functional L1s in the galago lineage (and presumably also in the slow loris). The prosimian ORF1 sequences could be further subdivided into subfamilies. ORF1 sequences from both the galago and slow loris have a premature termination codon near the 3′ end, not shared by the other mammalian sequences, that shortens the open reading frame by 288 bp. An analysis of synonymous and nonsynonymous substitutions for the 5′ and 3′ portions, that included intra- and inter-subfamily comparisons, as well as comparisons among the other mammalian sequences, suggested that this premature stop codon is a prosimian acquisition that has rendered the 3′ portion of ORF1 in these primates noncoding. Presented at the NATO Advanced Research Workshop onGenome Organization and Evolution, Spetsai, Greece, 16–22 September 1992     

Antibody estimation by indirect complement fixation test for foot-and-mouth disease in cattle.

Antibody against foot-and-mouth disease (FMD) virus was measured by the indirect complement fixation (ICF) test. For this test serum samples were collected from cattle experimentally infected with FMD virus of O, A and Asia 1 types, as well as cattle infected in the field. Two types of antigen were used. One was antigen derived from infected lingual epithelial culture prepared by Frenkel's method with each type of the virus. The other was antigen derived from the lingual epithelium of cattle infected by virus inoculation. ICF antibody began to be dectected about 4 5 days after inoculation. It reached a maximum titer 10 14 days after inoculation, remaining at this titer for about a week or two, and then decreased gradually. It was, however, detectable even 63 days after inoculation. The rise and fall of ICF antibody was parallel with that of neutralizing antibody, although that antibody was always lower in titer than this. ICF antibody was detected type-specifically from cattle infected experimentally and naturally. These results indicated that the ICF test was available for the routine serological diagnosis and epizootiological investigation and research.     

Metabolic studies using recombinant escherichia coli cells producing rat mitochondrial CYP24 CYP24 can convert 1alpha,25-dihydroxyvitamin D3 to calcitroic acid.

Previously we expressed rat 25-hydroxyvitamin D3 24-hydroxylase (CYP24) cDNA in Escherichia coli JM109 and showed that CYP24 catalyses three-step monooxygenation towards 25-hydroxyvitamin D3 and 1alpha,25-dihydroxyvitamin D3 [Akiyoshi-Shibata, M., Sakaki, T., Ohyama, Y., Noshiro, M., Okuda, K. & Yabusaki, Y. (1994) Eur. J. Biochem. 224, 335-343]. In this study, we demonstrate further oxidation by CYP24 including four- and six-step monooxygenation towards 25-hydroxyvitamin D3 and 1alpha,25-dihydroxyvitamin D3, respectively. When the substrate 25-hydroxyvitamin D3 was added to a culture of recombinant E. coli, four metabolites, 24, 25-dihydroxyvitamin D3, 24-oxo-25-hydroxyvitamin D3, 24-oxo-23, 25-dihydroxyvitamin D3 and 24,25,26,27-tetranor-23-hydroxyvitamin D3 were observed. These results indicate that CYP24 catalyses at least four-step monooxygenation toward 25-hydroxyvitamin D3. Furthermore, in-vivo and in-vitro metabolic studies on 1alpha,25-dihydroxyvitamin D3 clearly indicated that CYP24 catalyses six-step monooxygenation to convert 1alpha,25-dihydroxyvitamin D3 into calcitroic acid which is known as a final metabolite of 1alpha,25-dihydroxyvitamin D3 for excretion in bile. These results strongly suggest that CYP24 is largely responsible for the metabolism of both 25-hydroxyvitamin D3 and 1alpha,25-dihydroxyvitamin D3.     

Cell specificity and properties of the C-3 epimerization of Vitamin D3 metabolites

It is well documented that Vitamin D3 metabolites and synthetic analogs are metabolized to their epimers of the hydroxyl group at C-3 of the A-ring. We investigated the C-3 epimerization of Vitamin D3 metabolites in various cultured cells and basic properties of the enzyme responsible for the C-3 epimerization. 1alpha,25-Dihydroxyvitamin D3 [1alpha,25(OH)2D3], 25-hydroxyvitamin D3 [25(OH)D3] and 24,25-dihydroxyvitamin D3 [24,25(OH)2D3] were metabolized to the respective C-3 epimers in UMR-106 (rat osteosarcoma), MG-63 (human osteosarcoma), Caco-2 (human colon adenocarcinoma), LLC-PK1 (porcine kidney) and HepG2 (human hepatoblastoma)] cells, although the differences existed in the amount of each C-3 epimer formed with different cell types. In terms of maximum velocity (Vmax) and Michaelis constant (Km) values for the C-3 epimerization in microsome fraction of UMR-106 cells, 25(OH)D3 exhibited the highest specificity for the C-3 epimerization among 1alpha,25(OH)2D3, 25(OH)D3 and 24,25(OH)2D3. C-3 epimerization activity was not inhibited by various cytochrome P450 inhibitors and antiserum against NADPH cytochrome P450 reductase. Neither CYP24, CYP27A1, CYP27B1 nor 3(alpha --> beta) -hydroxysteroid epimerase (HSE) catalyzed the C-3 epimerization in vitro. Based on these results, the enzyme responsible for the C-3 epimerization of Vitamin D3 are thought to be different from already-known cytochrome P450-related Vitamin D metabolic enzymes and HSE.     

Identification of the amino acid residue of CYP27B1 responsible for binding of 25-hydroxyvitamin D3 whose mutation causes vitamin D-dependent rickets type 1

We previously reported the three-dimensional structure of human CYP27B1 (25-hydroxyvitamin D3 1alpha-hydroxylase) constructed by homology modeling. Using the three-dimensional model we studied the docking of the substrate, 25-hydroxyvitamin D3, into the substrate binding pocket of CYP27B1. In this study, we focused on the amino acid residues whose point mutations cause vitamin D-dependent rickets type 1, especially unconserved residues among mitochondrial CYPs such as Gln65 and Thr409. Recently, we successfully overexpressed mouse CYP27B1 by using a GroEL/ES co-expression system. In a mutation study of mouse CYP27B1 that included spectroscopic analysis, we concluded that in a 1alpha-hydroxylation process, Ser408 of mouse CYP27B1 corresponding to Thr409 of human CYP27B1 forms a hydrogen bond with the 25-hydroxyl group of 25-hydroxyvitamin D3. This is the first report that shows a critical amino acid residue recognizing the 25-hydroxyl group of the vitamin D3.     

A novel superoxide-producing NAD(P)H oxidase in kidney

During phagocytosis, gp91(phox), the catalytic subunit of the phagocyte NADPH oxidase, becomes activated to produce superoxide, a precursor of microbicidal oxidants. Currently increasing evidence suggests that nonphagocytic cells contain similar superoxide-producing oxidases, which are proposed to play crucial roles in various events such as cell proliferation and oxygen sensing for erythropoiesis. Here we describe the cloning of human cDNA that encodes a novel NAD(P)H oxidase, designated NOX4. The NOX4 protein of 578 amino acids exhibits 39% identity to gp91(phox) with special conservation in membrane-spanning regions and binding sites for heme, FAD, and NAD(P)H, indicative of its function as a superoxide-producing NAD(P)H oxidase. The membrane fraction of kidney-derived human embryonic kidney (HEK) 293 cells, expressing NOX4, exhibits NADH- and NADPH-dependent superoxide-producing activities, both of which are inhibited by diphenylene iodonium, an agent known to block oxygen sensing, and decreased in cells expressing antisense NOX4 mRNA. The human NOX4 gene, comprising 18 exons, is located on chromosome 11q14.2-q21, and its expression is almost exclusively restricted to adult and fetal kidneys. In human renal cortex, high amounts of the NOX4 protein are present in distal tubular cells, which reside near erythropoietin-producing cells. In addition, overexpression of NOX4 in cultured cells leads to increased superoxide production and decreased rate of growth. The present findings thus suggest that the novel NAD(P)H oxidase NOX4 may serve as an oxygen sensor and/or a regulator of cell growth in kidney.     

Multiplex polymerase chain reaction (PCR) with color-tagged module-shuffling primers for comparing gene expression levels in various cells

A method based on the multiplex polymerase chain reaction (PCR) and gel electrophoresis for the comparative analysis of gene expression levels was developed. Using the method many cDNA fragments from different sources can be compared simultaneously. Competitive PCR amplification of expressed genes from different sources was performed by using ‘module-shuffling primers’ (MPSs). The MPSs (labeled with different fluorophores) consist of sequence modules of 3 or 4 nt. The modules are arranged in different orders in each primer; therefore, the base sequences of the primers are different but their melting temperatures are identical. The genes expressed in different sources are ligated with tags complementary with the MPSs. Tag-ligated fragments are mixed in one tube and amplified at the same amplification efficiency by the MPSs. Amplified fragments are detected separately by multiple-color gel electrophoresis. This method can detect different amounts of each expressed gene, up to a difference in amounts of 30%, and its detection limit is 0.1 amol per assay.