Searching by Differential Display for New Genes Implicated in Induced Disease Resistance of Cucumber Treated with Acibenzolar-S-methyl

Acibenzolar-S-methyl (ASM) is a chemical activator of systemic disease resistance in plants. In this study, we used differential display to identify ASM-inducible defense response genes involved in induced disease resistance. As a result, we cloned three ASM-inducible genes from cucumber, encoding a chitinase, a putative protein disulfide isomerase and a putative mitochondrial-protein-like protein. Expression of these genes was induced within 24 hr after treatment of cucumber leaves with ASM. These results suggest that differential display is a useful tool for understanding the mode of action of ASM and defense responses. Received 6 September 2000/ Accepted in revised form 11 April 2001     

Gene expression profiles of CD4/CD8 double‐positive T cells in porcine peripheral blood

A characteristic subset of T cells, known as double positive T cells (DPTC) and expressing both cluster of differentiation 4 (CD4) and CD8, is observed in porcine peripheral blood. Previous studies suggested that DPTC might be memory cells. However, detailed phenotypes and functions of DPTC are yet to be fully elucidated and thus, the relatedness of DPTC with memory phenotypes remains unclear. In this study, DPTC gene expression profiles in peripheral blood were analyzed by DNA microarray in Experiment 1 and compared with those of CD4 single positive T cells (4SPTC) and CD8 single positive T cells (8SPTC). Expressions of IFNG, CCL5, NCK2, CCR2 and ITGB1 were higher than that of 4SPTC and 8SPTC. In contrast, expressions of CCR7 and SELL were lower than that of 4SPTC and 8SPTC. These results suggested that DPTC were either effector T cells or effector memory T cells (T_EM). Next, to determine whether DPTC were effector T cells or T_EM, differences in the response of DPTC and 8SPTC against immunized/primed antigens were compared (Experiment 2). While DPTC showed quick elevation of IL2 and CD25 gene expressions against in vitro stimulation of primed/immunized antigens, 8SPTC did not. These results suggest that at least some DPTC likely belong to T_EM.     

Effects of hairy vetch foliage application on nodulation and nitrogen fixation in soybean cultivated in three soil types

We investigated the effects of applying hairy vetch foliage on nodulation and atmospheric nitrogen (N₂) fixation in soybean cultivated in three soil types in pot experiments. Soybean plants were grown in Gley Lowland soil (GLS), Non-allophanic Andosol (NAS), and Sand-dune Regosol (SDR) with hairy vetch foliage application in a greenhouse for 45 days. In GLS, the nodule number was not influenced by the application, however, nodule dry weight and N₂ fixation activity tended to increase. In NAS and SDR, nodule formation was depressed by foliage application. Soybean plant growth was promoted in GLS and SDR but not in NAS. These promotive effects of hairy vetch foliage application on soybean plant growth in GLS were considered to be mainly caused by the increase in N₂ fixation activity of the nodules, whereas it was considered to be mainly caused by the increase in nitrogen uptake activity of the roots in SDR. The varying effects of hairy vetch foliage application on soybean nodulation may be due to soil chemical properties such as pH and cation exchange capacity, which are related to soil texture. Therefore, we conclude that it is important to use hairy vetch for soybean cultivation based on the different effects of hairy vetch on soybean plant growth in different soil types.     

Tetrahydrofolate-dependent vanillate and syringateO-demethylation links tightly to one-carbon metabolic pathway associated with amino acid synthesis and DNA methylation in the lignin metabolism ofSphingomonas paucimobilis SYK-6

Sphingomonas paucimobilis SYK-6 strain can degrade various lignin-related compounds. In the lignin metabolic pathway of this bacterium, vanillate and syringate are demethylated by the tetrahydrofolate (THF)-dependentO-demethylation system, which requires the enzymatic function of LigH. Upstream of theligH gene is the 5,10-methylene-THF reductase gene. Its gene product was essential for one-carbon metabolism involved in the amino acid synthesis and DNA methylation in all organisms. When themetF gene was inactivated in the genome of SYK-6, the resultant mutant, DLmetF, could not grow on vanillate and syringate as a sole carbon source. Furthermore, DLmetF showed significant accumulation of methyl-THF as a result of vanillate and syringateO-demethylation. We report here that THF-dependent vanillate and syringateO-demethylation links tightly to the one-carbon metabolic pathway that is associated with amino acid synthesis and DNA methylation, and the methyl group is the sole one-carbon source inS. paucimobilis SYK-6.     

Specific degradation ofβ-aryl ether linkage in synthetic lignin (dehydrogenative polymerizate) by bacterial enzymes ofSphingomonas paucimobilis SYK-6 produced in recombinantEscherichia coli

Sphingomonas paucimobilis SYK-6 produces unique and specific enzymes, such as-etherases,O-demethylases, and ring fission dioxygenases, for lignin degradation. Cleavage of arylglycerol--aryl ether linkage is the most important process in the lignin metabolic pathway ofS. paucimobilis SYK-6. We reported the genes (ligD, ligE, ligF) for enzymes that cleaved-aryl ether linkage of dimeric compounds in previous studies. In this study we synthesized the fluorescent high-molecular-weight lignin (UBE-DHP) by dehydrogenative polymerization. We investigated the-aryl ether cleavage ability of these enzymes produced in recombinantEscherichia coli. When UBE-DHP was incubated with LigF, 4-methylumbeliferone was released as a result of-aryl ether cleavage of O-methylumbelliferyl--hydroxypropiovanillone (compound III) incorporated in UBE-DHP. Here, we report that-etherase ofS. paucimobilis SYK-6 can be expressed inE. coli and is able to cleave the-aryl ether linkage in synthetic high-molecular-weight lignin.     

Molecular characterization of Cryptosporidium parvum detected in Japanese black and Holstein calves in Iwate Prefecture and Tanegashima Island,Kagoshima Prefecture,Japan

Cryptosporidium oocysts were found in 43 out of 77 calves from two farms in Iwate Prefecture and nine farms on Tanegashima Island, Kagoshima Prefecture, Japan. The DNA fragments of 18S ribosomal RNA (18S rRNA) gene were amplified by a nested PCR from 43 oocyst-positive as well as one oocyst-negative samples. All of them were precisely identified as C. parvum by analyzing the nucleotide sequences of the 18S rRNA gene. C. parvum oocyst-positive calves ranged in age from 6 to 13 days old and significantly have watery diarrhea (P<0.05). Sequences of the gene encoding the 60-kDa glycoprotein (GP60) in 43 Cryptosporidium oocyst-positive samples were identical to that of the zoonotic IIaA15G2R1 subtype. We therefore suggest that calves could be potential sources of C. parvum infections in humans.     

Decaying toxic wood as sodium supplement for herbivorous mammals in Gabon

African rainforest harbors herbivores at high density. However, because plants and soils typically lack in some essential minerals, rainforest is not always a suitable habitat for herbivores. How they fulfill the mineral requirements is therefore an important question to animal ecology and conservation. Although large marshes, called ‘bais’, are often mentioned as efficient mineral-resource, little information on other sodium resources has still been available. Our laboratory works and field surveys found that a peculiar item, decaying wood stumps of Anthostema aubryanum, played as a major sodium resource for herbivores in Moukalaba-Doudou National Park, Gabon. When A. aubryanum is alive, the sodium content of its bark is low and its latex is toxic. Sodium is accumulated in decaying stumps (mean=1,343 mg/kg dry matter). Eight herbivores visited stumps to ingest the dead wood. Fecal sample analysis revealed that western lowland gorillas, a species most-frequently using the stumps, consumed large amount of the dead wood as regular food. Our findings suggest that decaying A. aubryanum is critical sodium-resources and is a key species for herbivores in our study area. Importance of the A. aubryanum may be particularly large there, because it is a limited sodium-rich material that is available year round. Our study site is known as the site where the densities of several herbivores are among the highest at Central Africa. The relatively high herbivores density in our study site may partly depend on decaying A. aubryanum as sodium resources.     

Serological evidence of influenza A virus infection in Kuril harbor seals (Phoca vitulina stejnegeri) of Hokkaido, Japan

For proper management and conservation of the Kuril harbor seal (Phoca vitulina stejnegeri) through disease control, serological analysis was performed for influenza A virus infection in free-ranging seals in Hokkaido, Japan. Serum samples were collected from seals at Nosappu (231 seals), Akkeshi (16) and Erimo (75), between 1998 and 2005, and were analyzed by ELISA. Antibodies to the influenza A virus were detected only in seals from Nosappu. The incidences were 11% (1/9), 3% (2/66), 12% (7/59) and 6% (5/77) in 1998, 2003, 2004 and 2005, respectively. These suggest sporadic infection. Because antibody-positive seals included juvenile seals in each year, the infections were considered to have been circulated since no later than the late 1990s until recent years. ELISA-positive sera were analyzed by hemagglutination inhibition (HI) tests to determine the subtypes. Antibodies to the H3 and H6 subtypes were detected in 10 and 2 sera, respectively. Two of the sera that had antibodies to the H6 subtype also had antibodies to the H3 subtype. These two seals were considered to have been infected with both the H3 and H6 subtypes. This is the first investigation to find antibodies to the H6 subtype in seals. Although the H6 subtype had been isolated only from avians, genetic analysis had suggested that the H6 subtype could become a novel mammalian pathogen. For definitive diagnosis, detection of the virus from the tissue or mucus of seals is required.     

Antioxidative functions of natto,a kind of fermented soybeans: effect on LDL oxidation and lipid metabolism in cholesterol-fed rats

Natto water-soluble fractions, low-molecular-weight viscous substance, and soybean water extract, which had an inhibitory effect on the oxidation of low-density lipoproteins (LDL) in vitro, were fed to rats for 3 weeks. These fractions had no influence on the growth of rats, which were fed a basal diet containing 1% cholesterol, but lowered plasma triglyceride and total cholesterol. Inhibition of copper-oxidation of plasma and LDL ex vivo, and a reduction in lipid peroxidation in liver and aorta in vivo, were also observed. The antioxidant enzymes were not induced in rats fed natto fraction diets. These results demonstrate that ingestion of the natto fractions led to inhibition of LDL oxidation, and that the fractions perform direct antioxidant action in the body. It is suggested that natto fractions might help to prevent arteriosclerosis, as they appear to reduce lipid peroxidation and improve lipid metabolism.     

Cloud Water Deposition to Forest Canopies of Cryptomeria Japonica at Mt.Rokko, Kobe, Japan

Cloud water deposition to canopies of Cryptomeria japonica at Mt.Rokko, Kobe, Japan, was estimated from throughfall measurements and fog water collections carried out during a full year. Annual cloud frequency was 11.5–15.5% and liquid water content (LWC) was 0.059 g/m³. Since cloud water deposition on to forest canopies was significantly correlated with the amount of fog water collected, the former parameter could be quantitatively derived from throughfall measurements. Annual cloud water deposition on to Cryptomeria canopies was 1420–2860 mm (Av. 2140 mm), corresponding to 90–180% (Av. 122%) of annual rainfall. The rate of deposition was higher at the mountain ridge and the forest edge than at the mountain side and the forest interior. Annual deposition of SO₄ ^2?, NO₃ ^?, H⁺ and NH₄ ⁺ from cloud water was estimated as 204, 153, 2.5 and 58 kg/ha, respectively, equivalent to 5.8–11.7 times the corresponding deposition via rain. The values are equal to, or exceed, the maximum deposition reported for Appalachian forests in the eastern United States. Multiple regression analyses indicate that cloud water deposition on to Cryptomeria canopies was significantly correlated with the following three parameters: cloud frequency, LWC, and wind speed. Thus, these three factors apparently control cloud water deposition on to forest canopies.