Phenotype and function of murine discrete Peyer's patch macrophage derived - dendritic cells

The phenotype and function of peritoneal cavity macrophage-derived dendritic cells (PEC-DC) was previously reported. In this study we have gone further in using our established culture system to generated discrete Peyer's patch dendritic cells (DPP-DC) from murine discrete Peyer's patch macrophages (DPP-M?), following stimulation with granulocyte macrophage colony stimulating factor (GM-CSF) plus interleukin 4 (IL-4) for 7 days. DPP-M? from murine small intestines were obtained by mechanical disruption of discrete Peyer's patches (DPP), followed by metrizamide density gradient centrifugation to remove Peyer's patch resident DC and debri, after which an overnight adherent step in tissue culture medium was carried out for macrophage enrichment. Characterization of the generated DPP-DC was carried out using well-established criteria of morphology, expression of membrane antigens and capacity for antigen presentation. Dendritic cells expressed DEC-205, F4/80 and CD34 at high levels, but exhibited very low CD11c levels. They were shown to present soluble protein antigen to CD3(+) spleen T cells. A comparison of the surface antigen expression in the progenitor DPP-M? population and the generated DPP-DC showed a significant decrease in MHC class II levels and a marked down regulation of the co-stimulatory molecule CD86 (B7-2). High expression of the haemopoietic progenitor marker CD34 indicates that the generated DC, possess a haemopoietic rather than myeloid origin. Taken together, these results may provide a better understanding of the complex network regulating mucosal immune responses.     

Effects of light quality on conidiophore formation of the melon powdery mildew pathogen <Emphasis Type="Italic">Podosphaera xanthii</Emphasis>

The lengths of conidiophores in fungal colonies of the melon powdery mildew pathogen Podosphaera xanthii Pollacci KMP-6 N cultured under greenhouse (natural) conditions differed markedly from those cultured in a growth chamber. We hypothesized that light wavelength was responsible for the differences in conidiophore length. In this study, we examined the effects of light-emitting diode (LED) irradiation (purple, blue, green, orange, and red light) and white light on colony development and conidiophore formation in KMP-6 N using a stereomicroscope and a high-fidelity digital microscope. Colonies on leaves were flat under greenhouse conditions and under red LED light irradiation but were stacked under growth chamber conditions and under purple, blue, green, and orange LED light irradiation. In addition, KMP-6 N formed catenated conidia comprising six conidia per conidiophore under greenhouse conditions and red light but more than seven conidia per conidiophore under growth chamber conditions and purple, blue, green, and orange light. Furthermore, almost none of the conidia on top of the conidiophores grown under blue light were fully constricted. Therefore, these fungi could not scatter their conidia and spread infection. This is the first report of the effects of LED lights on conidiophore formation in the melon powdery mildew fungus P. xanthii. The results provide insight into the mechanisms underlying the responses of conidiophores to light of specific wavelengths and conidial scatter from conidiophores of melon powdery mildew fungi.     

In Vitro Suppression of Mycelial Growth of Fusarium oxysporum by Extracellular Chitosanase of Sphingobacterium multivorum and Cloning of the Chitosanase Gene csnSM1

A chitosan-degrading bacterium, isolated from field soil that had been amended with chitin, was identified as Sphingobacterium multivorum KST-009 on the basis of its bacteriological characteristics. The extracellular chitosanase (SM1) secreted by KST-009 was a 34-kDa protein and could be purified through ammonium sulfate precipitation, gel permeation column chromatography and SDS polyacrylamide gel electrophoresis. A chitosanase gene (csnSM1) was isolated from genomic DNA of the bacteria, and the entire nucleotide sequence of the gene and the partial N-terminal amino acid sequence of the purified SM1 were determined. The csnSM1 gene was found to encode 383 amino acids, 72 N-terminal amino acid residues were processed to produce the mature enzyme during the secretion process. Germinated microconidia of four formae speciales (lycopersici, radicis-lycopersici, melonis, and fragariae ) of Fusarium oxysporum were treated with SM1. Chitosanase treatment caused morphological changes, such as swelling of hyphal cells or indistinctness of hyphal cell tips and cessation or reduction of mycelial elongation. Received 2 May 2001/ Accepted in revised form 21 June 2001     

Bacteriological evaluation of composted manure solids prepared from anaerobic digested slurry for hygienic recycled bedding materials for dairy cows

Changes in mastitis‐causing pathogens, pH and water content in composted manure solids (CMS) prepared from digested slurry were evaluated during turning at 2‐day intervals for 8 days (C1–C4). The numbers of streptococci, coagulase‐negative staphylococci and coliforms were 2.6 × 10¹, 1.7 × 10² and 1.0 × 10¹ colony‐forming units (cfu)/g in CMS (C4) (summer), and these counts were markedly lower (P < 0.05) than those in CMS (C0 and C1). The bacterial counts ranged from 10¹ to 1.7 × 10² cfu/g in CMS (C4) (summer) and were within approved levels, <1 × 10⁶ cfu/g, indicating a minimal mastitis risk. The temperatures in CMS (C1–C4) increased to 63°C–74°C in summer and 67°C–70°C in winter. The mean pH values in CMS (C0–C4) were 9.2 in summer and 8.7 in winter, and water contents ranged from 61.7% to 69.6% in summer and 73.2% to 66.2% in winter. The significant decrease of pathogenic bacteria in CMS appears to be closely related to temperature >63°C for 8 days, pH 8.7–9.2, and water content 62% to 73%. This study demonstrates that prepared CMS has value as a recycled material with the potential to alleviate udder health issues in dairy cows.     

Screening of Dried Plant Seed Extracts for Adiponectin Production Activity and Tumor Necrosis Factor-Alpha Inhibitory Activity on 3T3-L1 Adipocytes

To search for dried plant seeds with potent anti-diabetes activity, we conducted a large scale screening for inhibitory activity on tumor necrosis factor-alpha and facilitating activity on adiponectin production in vitro. These activities in 3T3-L1 adipocytes were screened from ethanol extracts of 20 kinds of dried plant seed marketed in Japan. komatsuna (Brassica rapa var. perviridis), common bean (Phaseolus vulgaris L.), qing geng cai (Brassica rapa var. chinensis), green soybean (Glycine max), spinach (Spinacia oleracea L.) and sugar snap pea (Pisum sativum L.) markedly enhanced adiponectin production (11.3 ~ 12.7 ng/ml) but Japanese radish (Raphanus sativus), edible burdock (Arctium lappa L.), bitter melon (Momordica charantia) and broccoli (Brassica oleracea var. italica) did not (0.9 ~ 2.7 ng/ml). All adiponectin-production-enhancing seeds except spinach (2.7 pg/ml) and okra (Abelmoschus esculentus) (6.6 pg/ml) effectively decreased tumor necrosis factor-alpha levels (0.0 pg/ml). We further examined the effects on free radical scavenging activities in the dried seed extracts. Although scavenging activity correlated well with total phenolic content of samples, no correlation was observed with adiponectin production. These results point to the potential of dried seed extracts as a means to modify the activity of tumor necrosis factor-alpha for the adiponectin production.     

A dwarf bamboo (<Emphasis Type="Italic">Pleioblastus chino</Emphasis>) and winter browsing by Japanese hare (<Emphasis Type="Italic">Lepus brachyurus</Emphasis>) combine to limit establishment of transplanted native tree seedlings in an abandoned agricultural field

Natural forest recovery on abandoned farmland is hindered by a variety of factors and active restoration plays an important role when quick afforestation is desired. We investigated seedling survival of four transplanted native tree species (Quercus myrsinifolia, Quercus serrata, Aphananthe aspera, and Rhus sylvestris) by experimentally manipulating the vegetation cover, which was mainly dominated by dwarf bamboo (Pleioblastus chino), and herbivore access to the planting sites on farmland that had been abandoned for 15 years at the start of the study. Few transplanted seedlings of any species survived under intact vegetation cover, irrespective of herbivore presence. In gaps in the vegetation cover, winter browsing by Japanese hare (Lepus brachyurus) damaged all species. However, lower browsing frequency and higher resprouting ability after grazing of the seedlings enabled both Quercus species to survive better than the other species. These results indicate that dwarf bamboo and the hare jointly limit the establishment of native trees in old fields. If active afforestation by transplanting seedlings at sites dominated by dwarf bamboo is planned, a combination of vegetation removal, selection of suitable species, and temporary seedling protection will be most effective.     

Determination and Evaluation of Sampling Velocity in a Simple Passive Sampling Method to Monitor HNO3(g) in Ambient Air

We evaluated the applicability of a simple passive sampling method to quantify HNO₃(g) in ambient air. The method has the advantages of not only ease of operation but also low cost. A sampling velocity of 214 m day^?1 was determined based on the concentration of HNO₃(g) measured by the four-stage filter-pack method at nine sites located within a 250?×?250-km area in Japan. This sampling velocity was applied at sites located outside of area to verify the applicability and accuracy of the simple passive sampling method. The variation in the results for the application of the sampling velocity ranged from 0.39 to 0.95. The simple passive sampling method should be applied to sites with different meteorological conditions, and the obtained data should be used to obtain more significant information and/or to indicate the need for further developments in the methodology.     

Antioxidative activity of amino acids on tissue oxidative stress in human intestinal epithelial cell model

The protective effects of amino acids against H 2O 2-induced oxidative stress were investigated in an in vitro assay using human intestinal epithelial cells. Caco-2 cells were pretreated with amino acids (1, 2, and 5 mM) for 2 h and then stimulated with 1 mM H 2O 2 for 6 h. The secretion of IL-8, a proinflammatory mediator, was determined by ELISA as an indicator of tissue oxidative stress. The inhibition of H 2O 2-induced IL-8 secretion from Caco-2 cells was observed by pretreatment with Cys, Val, Ile, Leu, Trp, His, Lys, and Ala. Cys enhanced glutathione (GSH) biosynthesis enzyme activity and increased cellular GSH levels. Branched-chain amino acids such as Val, Ile, and Leu elevated activities of GSH S-transferase (GST) and catalase. Trp, His, and Lys caused increases in GST activity. Ala enhanced GSH reductase activity. These data suggest that specific amino acids exert protective effects against tissue oxidative stress in intestinal epithelial cells based on the structure.     

Simultaneous determination of all polyphenols in vegetables,fruits, and teas

Polyphenols, which have beneficial effects on health and occur ubiquitously in plant foods, are extremely diverse. We developed a method for simultaneously determining all the polyphenols in foodstuffs, using HPLC and a photodiode array to construct a library comprising retention times, spectra of aglycons, and respective calibration curves for 100 standard chemicals. The food was homogenized in liquid nitrogen, lyophilized, extracted with 90% methanol, and subjected to HPLC without hydrolysis. The recovery was 68-92%, and the variation in reproducibility ranged between 1 and 9%. The HPLC eluted polyphenols with good resolution within 95 min in the following order: simple polyphenols, catechins, anthocyanins, glycosides of flavones, flavonols, isoflavones and flavanones, their aglycons, anthraquinones, chalcones, and theaflavins. All the polyphenols in 63 vegetables, fruits, and teas were then examined in terms of content and class. The present method offers accuracy by avoiding the decomposition of polyphenols during hydrolysis, the ability to determine aglycons separately from glycosides, and information on simple polyphenol levels simultaneously.