Nitrite-reducing ability is related to growth inhibition by nitrite in Rhodobacter sphaeroides f. sp. denitrificans

Growth inhibition of Rhodobacter sphaeroides f. sp. denitrificans IL106 by nitrite under anaerobic-light conditions became less pronounced when the gene encoding nitrite reductase was deleted. Growth of another deletion mutant of the genes encoding nitric oxide reductase was severely suppressed by nitrite. Our results suggest that nitrite reductase increases the sensitivity to nitrite through the production of nitric oxide.     

A Functional Aqp1 Gene Product Localizes on The Contractile Vacuole Complex in Paramecium multimicronucleatum

In a ciliate Paramecium, the presence of water channels on the membrane of contractile vacuole has long been predicted by both morphological and physiological data, however, to date either the biochemical or the molecular biological data have not been provided. In the present study, to examine the presence of aquaporin in Paramecium, we carried out RT-PCR with degenerated primers designed based on the ParameciumDB, and an aquaporin cDNA (aquaporin 1, aqp1) with a full-length ORF encoding 251 amino acids was obtained from Paramecium multimicronucleatum by using RACE. The deduced amino acid sequence of AQP1 had NPA-NPG motifs, and the prediction of protein secondary structure by CNR5000 and hydropathy plot showed the presence of six putative transmembrane domains and five connecting loops. Phylogenetic analysis results showed that the amino acid sequence of AQP1 was close to that of the Super-aquaporin group. The AQP1-GFP fusion protein clearly demonstrated the subcellular localization of AQP1 on the contractile vacuole complex, except for the decorated spongiome membrane. The functional analyses of aqp1 were done by RNA interference-based gene silencing, using an established feeding method. The aqp1 was found to be crucial for the total fluid output of the cell, the function of contractile vacuole membranes.     

Spatial and elevational variation in fruiting phenology of a deciduous oak (Quercus crispula) and its effect on foraging behavior of the Asiatic black bear (Ursus thibetanus)

In habitats with elevational gradients, differences in the fruiting phenology of a single key food resource may affect the feeding behavior of an animal. The objectives of the present study were to assess (1) whether or not fruiting phenology and characteristics of Quercus crispula acorns differed with changes in altitude (900?C1,400?m asl) and area; (2) when bears foraged acorns in relation to their phenological development; and (3) where bears engaged in acorn foraging behavior with respect to acorn phenology. No difference in the fruiting phenology of acorns at various altitudes and locations was found, with the exception of changes in color and abscission period. Acorn abscission period occurred later at a site with an elevation of 1,200?m in Tochigi and at another with an elevation of 1,400?m in Gunma, making the available energy of acorns in the tree canopy (AET) greater and available for a longer time period at those two sites. Foraging of acorns by bears was observed at sites of moderate to high altitude between late September and mid-October. A threshold date when acorns became suitable for foraging by bears could not be identified, as the size and nutritional value of acorns increased continuously. Foraging activity of bears observed at moderate and high altitude sites corresponded with locations where AET was available in greater amounts and for a longer period of time for some sites; however, the small sample size precluded accurate assessment.     

Automated cell isolation from photodegradable hydrogel based on fluorescence image analysis

We report an automated cell-isolation system based on fluorescence image analysis of cell aggregates cultured in a photodegradable hydrogel. The system incorporates cell culture in a humidified atmosphere with controlled CO₂ concentration and temperature, image acquisition and analysis, micropatterned light exposure, and cell collection by pipetting. Cell aggregates were cultured on hydrogels, and target cells were selected by phase contrast and fluorescence image analysis. After degradation of the hydrogel by exposure to micropatterned UV light, cell aggregates were transferred to a collection vessel by robotic pipetting. We assessed the system for hydrogel degradation, recovery of target cells, and contamination by off-target cells. We demonstrated two practical applications of our method: (i) in cell aggregates from MCF-7-RFP strains in which 18.8% of cells produced red fluorescent protein (RFP), we successfully obtained 14 proliferative fluorescence-positive cell aggregates from 31-wells, and all of the isolated strains produced a higher proportion of RFP production than the original populations; (ii) after fluorescent immunostaining of human epidermal growth factor receptor 2 (HER2) in cancer cells, we successfully isolated HER2-positive cells from a mixed population of HER2-positive and -negative cells, and gene sequence analysis confirmed that the isolated cells mainly contained the target cells.     

Modulation of IgE synthesis by IgE-binding factors released by T cells of asthmatic patients with elevated serum IgE

The culture supernatants of unstimulated T cells (TCS) from asthmatic patients with elevated serum IgE were tested for IgE-binding factors (IgE-BFs) displaying the IgE-potentiating activity. The IgE-BFs were detected by their ability to inhibit the rosetting of RPMI 8866 cells with ox erythrocytes coupled with mouse monoclonal antibody (E-Mab) specific to Fc receptors for IgE (Fc epsilon R). TCS showing the rosette-inhibiting activity significantly enhanced the spontaneous IgE synthesis by B cells of allergic individuals. Interestingly, rosette-inhibiting factors could be removed by absorption with IgE-Sepharose from which they were subsequently eluated with acid buffer, indicating that the rosette inhibition was indeed mediated by IgE-BFs. In addition, such IgE-BFs had affinity for concanavalin A and lost their IgE-potentiating activity after treatment with trypsin and neuraminidase. In contrast, T cells treated with tunicamycin released IgE-suppressing factors capable of inhibiting the IgE-potentiating activity of TCS derived from untreated T cells. On the other hand, the culture supernatants from subpopulations depleted of Fc epsilon R+ T cells but not of Fc gamma R+ T cells contained neither rosette-inhibiting factors nor IgE-potentiating factors, suggesting that IgE-BFs were released by in vivo pre-activated Fc epsilon R+ T cells. With regard to circulating Fc epsilon R+ T cells determined by E-Mab, they were significantly higher in asthmatic patients with elevated serum IgE (0.77 +/- 0.15%) than in normal subjects (0.17 +/- 0.07%) in spite of a very small proportion of T cells bearing Fc epsilon R.     

Allergen induced histamine release and immunoreactive-leukotriene C4 generation from leukocytes in mite sensitive asthmatic patients

Leukocytes from mite sensitive asthmatic patient were challenged with the allergen and the supernatant was assayed for histamine and immunoreactive-leukotriene C4 (i-LTC4). The release of histamine was quantitated by an automated fluorometric technique and i-LTC4 was determined using a commercial radioimmunoassay kit. The results of analysis of the supernatant by high speed liquid chromatography, together with observations of modulation of the formation by agents, indicated that i-LTC4 consisted of LTC4 with a little amount of LTD4. i-LTC4 was generated as a result of basophil activation but not derived from the other cells such as monocytes and eosinophils. Allergen induced a concentration-dependent release of histamine and i-LTC4 and the maximal release of histamine and i-LTC4 occurred at the same dose of the allergen. At optimal concentration of the allergen, basophils produced 20.4 +/- 17.9 ng of i-LTC4/10(6)-cells (mean +/- S.D., n = 39) and histamine release was 55.6 +/- 20.1% of total histamine. There was a significant correlation in the capacity of leukocytes to release histamine and i-LTC4 (r = 0.47, p less than 0.01). We found a correlation between maximal histamine release or cell sensitivity, allergen concentration for 50% histamine release, and a ratio of specific IgE to mite to total IgE in the serum, but the amount of i-LTC4 failed to correlate significantly with the ratio. The releasability and the cell sensitivity of asthmatic patients' cells to the allergen for histamine release paralleled the severity to symptoms, but this correlation was not significant in i-LTC4 generation.     

Sulfated vizantin inhibits biofilm maturation by Streptococcus mutans

Streptococcus mutans is the main pathogen of dental caries and adheres to the tooth surface via soluble and insoluble glucans produced by the bacterial glucosyltransferase enzyme. Thus, the S. mutans glucosyltransferase is an important virulence factor for this cariogenic bacterium. Sulfated vizantin effectively inhibits biofilm formation by S. mutans without affecting its growth. In this study, less S. mutans biofilm formation occurred on hydroxyapatite discs coated with sulfated vizantin than on noncoated discs. Sulfated vizantin showed no cytotoxicity against the human gingival cell line Ca9-22. Sulfated vizantin dose-dependently inhibited the extracellular release of cell-free glucosyltransferase from S. mutans and enhanced the accumulation of cell-associated glucosyltransferase, compared with that observed with untreated bacteria. Sulfated vizantin disrupted the localization balance between cell-associated glucosyltransferase and cell-free glucosyltransferase, resulting in inhibited biofilm maturation. These results indicate that sulfated vizantin can potentially serve as a novel agent for preventing dental caries.