Fern gametophytes of Angiopteris lygodiifolia and Osmunda japonica harbor diverse Mucoromycotina fungi

Journal of Plant Research - Mycorrhizal symbiosis between plants and fungi is ubiquitous, and has been played key roles in plant terrestrialization and diversification. Although arbuscular...     

Determination of human plasma xanthine oxidase activity by high-performance liquid chromatography

An assay for human plasma xanthine oxidase activity was developed with pterin as the substrate and the separation of product (isoxanthopterin) by high-performance liquid chromatography with a fluorescence detector. The reaction mixture consists of 60 μl of plasma and 240 μl of 0.2 M Tris-HCl buffer (pH 9.0) containing 113 μM pterin. With this assay, the activity of plasma xanthine oxidase could be easily determined despite its low activity. As a result, it could be demonstrated that the intravenous administration of heparin or the oral administration of ethanol did not increase plasma xanthine oxidase activity in normal subjects, and also that plasma xanthine oxidase activity was higher in patients with hepatitis C virus infection than in healthy subjects or patients with gout. In addition, a single patient with von Gierke's disease showed a marked increase in the plasma activity of this enzyme, relative to that apparent in normal subjects.     

Cutting edge: Role of Toll-like receptor 9 in CpG DNA-induced activation of human cells

Unmethylated CpG motifs present in bacterial DNA stimulate a rapid and robust innate immune response. Human cell lines and PBMC that recognize CpG DNA express membrane-bound human Toll-like receptor 9 (hTLR9). Cells that are not responsive to CpG DNA become responsive when transfected with hTLR9. Expression of hTLR9 dramatically increases uptake of CpG (but not control) DNA into endocytic vesicles. Upon cell stimulation, hTLR9 and CpG DNA are found in the same endocytic vesicles. Cells expressing hTLR9 are stimulated by CpG motifs that are active in primates but not rodents, suggesting that evolutionary divergence between TLR9 molecules underlies species-specific differences in the recognition of bacterial DNA. These findings indicate that hTLR9 plays a critical role in the CpG DNA-mediated activation of human cells.     

Microsomal carbonyl reductase responsible for reduction of 4-phenyl-3-buten-2-one in rats

trans-4-Phenyl-3-buten-2-one (PBO), a flavoring additive, was transformed to the carbonyl-reduced product, trans-4-phenyl-3-buten-2-ol (PBOL) by rat liver microsomes, but not by liver cytosol, in the presence of NADH or NADPH. PBOL formed was identified by comparison with an authentic sample. The reductase activity was not inhibited by quercitrin, an inhibitor of cytosolic carbonyl reductase. The carbonyl reduction product of PBO by liver microsomes was identified as the R-enantiomer of PBOL by HPLC analysis. Rat blood also exhibited the carbonyl reductase activity in the presence of NADH or NADPH, but to a lesser extent.     

Structural requirements of a human deoxyribonuclease II for the development of the active enzyme form, revealed by site-directed mutagenesis

Using site-directed mutagenesis, we eliminated three potential N-glycosylation sites (N86, N212, and N266) of human deoxyribonuclease II (DNase II), conserved in mammalian enzymes, and a proteolytic processing site (Q46-R47), forming a propeptide subunit of the enzyme. We expressed a series of these mutant DNase II constructs in COS-7 and Hep G2 cells. Liberation of each glycosylation site at N86 and N266 and the cleavage site interfered dramatically with expression of the intracellular and secreted DNase II activities, irrespective of cell line transfected. A chimeric mutant in which the signal peptide of the DNase II was replaced with that of human DNase I had no intracellular or secreted enzyme activity. Therefore, a simultaneous attachment of a carbohydrate moiety to N86 and N266, cleavage of the propeptide from the single DNase II precursor, and the inherent signal peptide might be required for subcellular sorting and proteolytic maturation of the enzyme.     

Genes encoding light-harvesting and reaction center proteins from Chromatium vinosum

Sequencing of a 3.4 kb DNA fragment isolated from the photosynthetic purple sulfur bacterium Chromatium vinosum and of PCR products has resulted in identification of the Chr. vinosum pufL, pufM, and pufC genes, reading from the 5 to the 3 direction, and coding, respectively, for the L, M and cytochrome c subunits of the reaction center of this bacterium. Other PCR products have been used to obtain complete sequences for the pufB and pufA genes, located immediately upstream from pufL and encoding the apoproteins of two Chr. vinosum light- harvesting proteins. The 3-portion of the bchZ gene, a gene that codes for a protein involved in the biosynthesis of bacteriochlorophyll, has been located immediately upstream from pufB. A second pufB gene, pufB2, has been located downstream from pufC, as has the 5-portion of a second pufA gene, pufA2. The location of a second set of pufB and pufA genes, encoding light- harvesting proteins, downstream from pufC has not previously been reported for any photosynthetic bacterium. Translation of the gene sequences encoding these Chr. vinosum light-harvesting proteins reveals both similarities to and differences from the amino acid sequences, obtained from direct sequencing of the apoproteins, previously reported for Chr. vinosum light-harvesting proteins. Translation of these gene sequences, and of those for pufL, pufM and pufC, revealed significant homology, at the amino acid level, to the corresponding peptides of photosynthetic purple non-sulfur bacteria.     

In vivo monitoring of hydroxyl radical generation caused by x-ray irradiation of rats using the spin trapping/EPR technique

Measurement of hydroxyl radical (*OH) in living animals irradiated with ionizing radiation should be required to clarify the mechanisms of radiation injury and the in vivo assessment of radiation protectors, because generation of *OH is believed to be one of the major triggers of radiation injury. In this study, *OH generation was monitored by spin trapping the secondary methyl radical formed by the reaction of *OH with dimethyl sulfoxide (DMSO). Rats were injected intraperitoneally with a DMSO solution of alpha-phenyl-N-tert-butylnitrone (PBN). X-irradiation of the rats remarkedly increased the six-line EPR signal in the bile. The strengthened signal was detectable above 40 Gy. Use of 13C-substituted DMSO revealed that the signal included the methyl radical adduct of PBN as a major component. The EPR signal of the PBN-methyl radical adduct was completely suppressed by preadministration of methyl gallate, a scavenger of *OH but not of methyl radical. Methyl gallate did not reduce the spin adducts to EPR-silent forms. These observations indicate that what we were measuring was *OH generated in vivo by x-irradiation. This is the first report of the in vivo monitoring of *OH generation at a radiation dose close to what people might receive in the case of radiological accident or radiation therapy.