Shigenori Sonoki

Fungal bioconversion of toxic polychlorinated biphenyls by white-rot fungus, Phlebia brevispora

Toxic coplanar polychlorinated biphenyls (Co-PCBs) were used as substrates for a degradation experiment with white-rot fungus, Phlebia brevispora TMIC33929, which is capable of degrading polychlorinated dibenzo-p-dioxins. Eleven PCB congener mixtures (7 mono-ortho- and 4 non-ortho-PCBs) were added to the cultures of P. brevispora and monitored by high resolution gas chromatography and mass spectrometry (HRGC/HRMS). Five PCB congeners, 3,3′,4,4′-tetrachlorobiphenyl, 2,3,3′,4,4′-pentachlorobiphenyl, 2,3′,4,4′,5-pentachlorobiphenyl, 3,3′,4,4′,5-pentachlorobiphenyl, and 2,3′,4,4′,5,5′-hexachlorobiphenyl were degraded by P. brevispora. To investigate the fungal metabolism of PCB, each Co-PCB was treated separately by P. brevispora and the metabolites were analyzed by gas chromatography and mass spectrometry (GC/MS) and identified on the basis of the GC/MS comparison with the authentic compound. Meta-methoxylated metabolite was detected from the culture containing each compound. Additionally, para-dechlorinated and -methoxylated metabolite was also detected from the culture with 2,3,3′,4,4′-pentachlorobiphenyl, 2,3′,4,4′,5-pentachlorobiphenyl, and 2,3′,4,4′,5,5′-hexachlorobiphenyl, which are mono-ortho-PCBs. In this paper, we identified the congener specific degradation of coplanar PCBs by P. brevispora, and clearly proved for the first time by identifying the metabolites that the white-rot fungus, P. brevispora, transformed recalcitrant coplanar PCBs.

Metabolism of hydroxylated PCB congeners by cloned laccase isoforms.

The white-rot fungus T. versicolor UAMH 8272 produced two groups of laccases, each of which included several isoforms showing different isoelectric points (pI). Group 1 and group 2 laccases, respectively, displayed higher pI 5–6 and lower pI 3–4. Of the four cloned full-length laccase cDNAs, Lac 1 and Lac 4 were expressed in the heterologous protein expression system using Aspergillus oryzae. The measured pI of each Lac 1 and Lac 4 expressed in A. oryzae was lower than that of pI predicted from the amino acid composition. With this regard, isoelectric focusing of Lac 1 showed the presence of multiple protein bands in the 3.0–4.0 pI range, although the predicted pI value of Lac 1 was 4.7. Similarly, Lac 4 exhibited a pI value which was lower than that predicted (3.6 vs. 4.3, respectively). In all tested hydroxyPCBs, higher chlorinated hydroxyPCBs were less susceptible to in vitro degradation by laccase than lower chlorinated hydroxyPCBs. Although Lac 4 showed a generally higher activity than Lac 1, the two laccases were characterized by quite different substrate specificity toward two hydroxy-tetrachlorobiphenyl congeners. Two metabolites were obtained from the metabolism of hydroxy-pentachlorobiphenyl: a ten chlorine-substituted dimer with a C–O bond, and one with a C–C bond.

High-performance liquid chromatographic analysis of fluorescent derivatives of adenine and adenosine and its nucleotides: optimization of derivatization with chloroacetaldehyde and chromatographic procedures

Abstract The use of chloroacetaldehyde (CAA) as a potential precolumn fluorimetric labelling reagent for adenine compounds was examined in detail. The reaction kinetics was greatly influenced by parameters such as the pH, temperature and CAA concentration. These parameters were optimized with regard to the reaction yield. The resulting procedure for CAA derivatization of adenine compounds was found to be excellent for quantitative analysis. Because the CAA derivatives of the adenine compounds studied were markedly stable, precise quantitative results were easily obtained using high-performance liquid chromatography. CAA derivatives of the five adenine compounds, i.e., 1,N6-etheno-adenine, -adenosine, -adenosine 5′-monophosphate, -adenosine 5′-diphosphate and -adenosine 5′-triphosphate were separated. The detection limits for the 1,N6-etheno derivatives were 0.5—1.7 pmol per 10-μl injection. Due to its simplicity, speed, sensitivity and selectivity, this procedure is recommended for use in studies on the metabolism and biology of adenine compounds.

Liquid chromatographic determination of 5-methylcytosine in DNA with fluorescence detection

Abstract A novel method to determine the content of 5-methylcytosine in DNA precisely, sensitively and simply has been developed using liquid chromatography (LC) with fluorescence detection. DNA was mildly digested by cooperation of nuclease P1 and DNase I, then the generated 2′-deoxynucleoside 5′-monophosphates (dNmp) were specifically derivatized fluorometrically at the position of the phosphoric acid moiety with 5-dimethylaminonaphthalene-1-[ N -(2-aminoethyl)]sulfonamide (dansylEDA), followed by the separation of fluorescent dansylEDA derivatives of dNmp on the reversed-phase partition column. The method was more than 10 times as sensitive as the conventional ultraviolet detection method.

Identification of cytochrome P450 monooxygenase genes from the white-rot fungus Phlebia brevispora.

Three cytochrome P450 monooxygenase (CYP) genes, designated pb-1, pb-2 and pb-3, were isolated from the white-rot fungus, Phlebia brevispora, using reverse transcription PCR with degenerate primers constructed based on the consensus amino acid sequence of eukaryotic CYPs in the O2-binding, meander and heme-binding regions. Individual full-length CYP cDNAs were cloned and sequenced, and the relative nucleotide sequence similarity of pb-1 (1788 bp), pb-2 (1881 bp) and pb-3 (1791 bp) was more than 58%. Alignment of the deduced amino acid (aa) sequences of pb-1-pb-3 showed that these three CYPs belong to the same family with > 40% aa sequence similarity, and pb-1 and pb-3 are in the same subfamily, with > 55% aa sequence similarity. Furthermore, pb-1-pb-3 appeared to be a subfamily of CYP63A (CYP63A1-CYP63A4), found in Phanerochaete chrysosporium. The phylogenetic tree constructed by 500 bootstrap replications using the neighbor-joining method showed that the evolutionary distance between pb-1 and pb-3 was shorter than that between pb-2 and pb-1 (or pb-3). Exon-intron analysis of pb-1 and pb-3 showed that both genes have nearly the same number, size and order of exons and the types of introns, also indicating both genes appear to be evolutionarily close. It is interesting that the transcription level of pb-3 was evidently increased above the pb-1 transcription level by exposure to 12 coplanar PCB congeners and 2,3,7,8-tetrachlorodibenzo-p-dioxin, though the two genes were evolutionarily close.

109 A BRIDGE OF SPERM TAIL BETWEEN BLASTOMERES ENHANCED PROTEIN MIGRATION IN THE RAT TWO-CELL STAGE EMBRYOS

The aim of the present study was to examine transient expression of transgene injected into nuclei of rat 2-cell stage embryos. We also investigated the relationship between expression in both blastomeres and tail position of penetrated spermatozoa in rat 2-cell stage embryos. Rat 2-cell stage embryos were recovered from superovulated Wistar females mated with same strain mature males at 48 h after hCG injection. DNA fragments, as the transgene containing the EGFP (enhanced green fluorescent protein) gene controlled under the CMV-IE promoter, were microinjected into one nucleus of 2-cell stage embryos. After microinjection, embryos were cultured in KRB at 37.0°C in a 5% CO2 and 95% humidified air until observation. First, transient EGFP expression in 151 injected embryos was observed using a fluorescence microscope at 6 h intervals until 48 h after injection. At 6 h after microinjection fluorescent embryos were detected, and the proportion of fluorescent embryos increased over time. The rate reached maximum (84%, 52/62) at 24 h after microinjection, and several fluorescent patterns of fluorescent blastomeres in the embryos were observed. There were blastomeres with the same or different fluorescence levels and a single fluorescent blastomere. Second, to assess tail position of the penetrated sperm in the fluorescent embryos, 75 whole mount specimens were observed by inverted phase-contrast microscopy at 24 h after the injection. Also, parthenogenetic 2-cell stage embryos that never contained sperm tail were microinjected with the transgene and observed in the same manner. To obtain parthenogenetic 2-cell embryos, 80 ovulated ova were collected from non-mated females, and incubated with 2 mM 6-DMAP for 4 h. The ova were additionally cultured for 20 h in KRB at 37.0°C in a 5% CO2 and 95% humidified air. In embryos with both blastomeres fluorescent (94%, 33/35), the sperm tail existed in both blastomeres like a bridge between blastomeres. In contrast, in one embryo with a single fluorescent blastomere (4%, 1/24), the sperm tail existed in both blastomeres, and in other embryos with a single fluorescent blastomere (75%, 18/24), the sperm tail was positioned in the one blastomere. On the other hand, in 63 parthenogenic rat 2-cell embryos in which there was no sperm tail, most embryos (86%, 54/63) had a single fluorescent blastomere at 24 h after microinjection. The results indicated that the sperm tail position in the 2-cell embryos makes the protein migration variable. In conclusion, when the CMV-IE/EGFP gene was microinjected into nuclei of rat 2-cell embryos, at 24 h after the microinjection the EGFP was detected in most embryos; however, fluorescent patterns in blastomeres varied. It seems that EGFP derived from the transgene injected into one blastomere may move into another blastomere in rat 2-cell stage embryos, and that the presence of a sperm tail in both blastomeres may influence EGFP distribution.

S19.19 Glycosphingolipids of plerocercoids of parasite,Spirometra erinacei

S19.17 Unusual Structures of Gangliosides from Starfishes G. P. Smirnova and N. V. Chekareva N. D. Zelinsky Institute of Organic Chemistry, Russian Academy of Sciences, Moscow, Russia. Gangliosides containing unusual oligiosaccharide chains were isolated from starfish Asterias rathbuni and Leptychaster anomalus belonging to different orders. Their structures were elucidated by chemical and physico-chemical methods. The main ganglioside from A. rathbuni was found to contain pentasaccharide chain with two 8-O-methyl-N-glycolylneuraminic acid residues bound to one N-acetylgalactosamine residue in positions 3 and 6:8-O-Me-NeuGca2-3 (8-O-MeNeuGca2-6)GalNAc/31-3Galfll-4Glcpl-lCer. The ganglioside from L. anomalus contained tetrasaccharide chain where Nglycolylneuraminic acid residue accupied the subterminal position and is glyeosylated at 0-4 by the terminal galactopyranose residue: Gal/31-4NeuGc2-3Gal/31-4Glcpl-lCer. The lipid moieties of both gangliosides were shown to contain normal and a-hydroxy fatty acids at a ratio of about 1:1. Sphingosines were detected in the L. anomalus ganglioside, whereas approximately equal amounts of sphingosines and phytosphingosines were found in the A. rathbuni ganglioside. Quantitative analysis of long-chain bases and fatty acids was carried out.

Studies on Harmful Gas Adsorbents Containing Chiefly Gas Adsorption Resins.

Two kinds of harmful gas (such as malodorous gas) adsorbent containing gas adsorption resins were developed. One is a polystyrene polyamine metal (Co) chelating resin capable of adsorbing more than 90% of sulfur-containing (C2H5SH and H2S etc.) or acidic gases (SOx, NOx and HCl etc.) within 60 minutes. The other is a polystyrene polysulfonic acid resin that can adsorb more than 90% of basic gases such as NH3 within 60 minutes. Thus, both resins have large gas adsorption capacity and their stability is good. The harmful gas adsorbents obtained by mixing both resins with active charcoal can adsorb a wide range of harmful gases. Both resins are very economical since they can be regenerated. The practical application of these resins are as follows. (1) Both can be used to remove specific harmful gases. (2) The harmful gas adsorbent is placed in a gas circulating device. (3) The gas adsorption sheet comprising of many corrugated layers of gas adsorbent can be hung from a wall. It should thus be possible to use these resins in many places such as lavatories, childcare rooms, hospitals, building and cars, factories and tunnels and be used to prevent corrosion of precision instruments.