Rat cytochrome P450-mediated transformation of dichlorodibenzo-p-dioxins by recombinant white-rot basidiomycete Coriolus hirsutus

Rat cytochrome P450, CYP1A1, has been reported to play an important role in the metabolism of mono-trichlorodibenzo-p-dioxins (M-TriCDDs). To breed lignin (and M-TetraCDDs)-degrading basidiomycete Coriolus hirsutus strains producing rat CYP1A1, an expression cassette [C. hirsutus gpd promoter-C. hirsutus gpd 5′ portion (224-bp of 1st exon–8th base of 4th exon)-rat cyp1a1 cDNA-Lentinula edodes priA terminator] was constructed and inserted into pUCR1 carrying the C. hirsutus arg1 gene. The resulting recombinant plasmid, MIp5-(cyp1a1 + arg1) was introduced into protoplasts of C. hirsutus monokaryotic strain OJ1078 (Arg⁻, Leu⁻), obtaining three good Arg⁺ transformants. These transformants [ChTF5-2(CYP1A1), ChTF5-4(CYP1A1), and ChTF5-6(CYP1A1)] were estimated to carry nine, six, and seven copies of the expression cassette on their chromosomes, respectively. Immunoblot analysis revealed that the three transformants produce similar amounts of rat CYP1A1 enzyme. ChTF5-2(CYP1A1), ChTF5-4(CYP1A1), ChTF5-6(CYP1A1) and recipient OJ1078 were cultivated in a liquid medium containing 2,7/2,8(at a ratio of 1:1)-dichlorodibenzo-p-dioxins (2,7/2,8-DCDDs) and the amount of intra- and extracellular 2,7/2,8-DCDDs remaining was measured. The results showed that all three transformants efficiently transform 2,7/2,8-DCDDs through the action of the recombinant rat CYP1A1 enzyme.     

An Inhibitory Role of Nitric Oxide in the Dynamic Regulation of the Blood-Brain Barrier Function

1. The present study aimed at elucidating the effect of nitric oxide (NO) on blood-brain barrier (BBB) function with mouse brain capillary endothelial (MBEC4) cells. 2. Histamine (20–100 μM) evoked NO production (1.6–7 μM) in MBEC4 cells in a dose-dependent manner. 3. The permeability coefficient of sodium fluorescein for MBEC4 cells and the cellular accumulation of rhodamine 123 in MBEC4 cells were increased dose-dependently by the addition of NO solutions (14 and 28 μM) every 10 min during a 30-min period. 4. The present study demonstrated that NO increased the permeability and inhibited the P-glycoprotein efflux pump of brain capillary endothelial cells, suggesting that NO plays an inhibitory role in the dynamic regulation of the BBB function.     

Structure of beta-glucan oligomer from laminarin and its effect on human monocytes to inhibit the proliferation of U937 cells

We analyzed the human monocyte-stimulating ability of laminarin from Eisenia bicyclis, lichenan from Cetraria islandica, and their oligomers depolymerized with endo-1,3-beta-glucanase from Arthrobacter sp. The respective beta-glucan oligomers with different degrees of polymerization (DP) were fractionated from hydrolytic products of laminarin and lichenan using gel-filtration chromatography. The monocyte-conditioned medium pre-cultured in the presence of a fraction of beta-glucan oligomer (DP>/=8) from laminarin exhibited inhibitory activity against the proliferation of human myeloid leukemia U937 cells, while those pre-cultured with other beta-glucan oligomers and the original laminarin and lichenan showed little or no activity. NMR analysis indicated that the beta-glucan oligomer (DP>/=8) has an average DP value of 13, and its ratio of beta-1,3- to beta-1,6-linkages in glucopyranose units was estimated to be 1.3:1. These results indicate that the beta-1,3-glucan oligomer with a higher content of beta-1,6-linkage stimulates monocytes to inhibit the proliferation of U937 cells.     

Predatory Potential of Freshwater Animals on an Invasive Agricultural Pest, the Apple Snail Pomacea canaliculata (Gastropoda: Ampullariidae), in Southern Japan

The apple snail Pomacea canaliculata is an invasive species and a serious pest of rice in many Asian countries. We studied predatory activities of various animals living in Japanese freshwater habitats, by keeping each individual of a potential predator species with 36 snails of various sizes for three days in the aquarium. Forty-six species were tested, and 26 in eight classes fed on small snails. A species of leech, crabs, the common carp, turtles, the mallard duck and the Norway rat attacked even adult snails of 20–30 mm in shell height. These findings will be helpful in identifying effective predators for biological control of the pest snail. In addition, most of the animals attacking snails are reported to be common in rivers or ponds, but few live in modernized paddy fields having little connections with natural water systems. This may be a reason why this snail maintains large populations in paddy fields but not in other freshwater habitats.     

R-state haemoglobin with low oxygen affinity: crystal structures of deoxy human and carbonmonoxy horse haemoglobin bound to the effector molecule L35

Although detailed crystal structures of haemoglobin (Hb) provide a clear understanding of the basic allosteric mechanism of the protein, and how this in turn controls oxygen affinity, recent experiments with artificial effector molecules have shown a far greater control of oxygen binding than with natural heterotropic effectors. Contrary to the established text-book view, these non-physiological compounds are able to reduce oxygen affinity very strongly without switching the protein to the T (tense) state. In an earlier paper we showed that bezafibrate (BZF) binds to a surface pocket on the alpha subunits of R state Hb, strongly reducing the oxygen affinity of this protein conformation. Here we report the crystallisation of Hb with L35, a related compound, and show that this binds to the central cavity of both R and T state Hb. The mechanism by which L35 reduces oxygen affinity is discussed, in relation to spectroscopic studies of effector binding.     

Dissection of the nuclear genome of barley by chromosome flow sorting

Isolation of mitotic chromosomes using flow cytometry is an attractive way to dissect nuclear genomes into their individual chromosomal components or portions of them. This approach is especially useful in plants with complex genomes, where it offers a targeted and hence economical approach to genome analysis and gene cloning. In several plant species, DNA of flow-sorted chromosomes has been used for isolation of molecular markers from specific genome regions, for physical mapping using polymerase chain reaction (PCR) and fluorescence in situ hybridization (FISH), for integration of genetic and physical maps and for construction of chromosome-specific DNA libraries, including those cloned in bacterial artificial chromosome vectors. Until now, chromosome analysis and sorting using flow cytometry (flow cytogenetics) has found little application in barley (2n = 14, 1C ∼ 5,100 Mbp) because of the impossibility of discriminating and sorting individual chromosomes, except for the smallest chromosome 1H and some translocation chromosomes with DNA content significantly different from the remaining chromosomes. In this work, we demonstrate that wheat–barley ditelosomic addition lines can be used to sort any arm of barley chromosomes 2H–7H. Thus, the barley genome can be dissected into fractions representing only about 6–12% of the total genome. This advance makes the flow cytogenetics an attractive tool, which may greatly facilitate genome analysis and gene cloning in barley.     

Hydrogen peroxide spraying alleviates drought stress in soybean plants

To ascertain the effect of exogenously applied hydrogen peroxide (H₂O₂) on drought stress, we examined whether the spraying of soybean leaves with H₂O₂ would alleviate the symptoms of drought stress. Pre-treatment by spraying leaves with H₂O₂ delayed foliar wilting caused by drought stress compared to leaves sprayed with distilled water (DW). Additionally, the relative water content of drought-stressed leaves pre-treated with H₂O₂ was higher than that of leaves pre-treated with DW. Therefore, we analyzed the effect of H₂O₂ spraying on photosynthetic parameters and on the biosynthesis of oligosaccharides related to water retention in leaves during drought stress. Under conditions of drought stress, the net photosynthetic rate and stomatal conductance of leaves pre-treated with H₂O₂ were higher than those of leaves pre-treated with DW. In contrast to DW spraying, H₂O₂ spraying immediately caused an increase in the mRNA levels of d-myo-inositol 3-phosphate synthase 2 (GmMIPS2) and galactinol synthase (GolS), which encode key enzymes for the biosynthesis of oligosaccharides known to help plants tolerate drought stress. In addition, the levels of myo-inositol and galactinol were higher in H₂O₂-treated leaves than in DW-treated leaves. These results indicated that H₂O₂ spraying enabled the soybean plant to avoid drought stress through the maintenance of leaf water content, and that this water retention was caused by the promotion of oligosaccharide biosynthesis rather than by rapid stomatal closure.