Automatic counting of FISH-labeled microbes by an LED illuminated detecting apparatus

ABSTRACT:   The Bioplorer (BP) (Matsushita Ecology Systems Co. Ltd, Kasugai, Aichi-ken, Japan) is an apparatus that consists of a light-emitting diode and optical and image analysis systems. This instrument has been developed and used for the semi-automatic counting of total microbes, mainly in food, cosmetics and industrial products. In this study, the applicability of BP to the detection and enumeration of eukaryotic and prokaryotic cells, after the fluorescent in situ hybridization (FISH) procedure, was examined. The cells of a yeast ( Candida albicans ) and a bacterium ( Escherichia coli ) were specifically labeled using an oligonucleotide probe with fluorochrome and then counterstained with 4', 6-diamidino-2-phenylindole (DAPI). The numbers of cells after FISH treatment and DAPI staining agreed well, indicating that the BP optical device and image analysis system had enough sensitivity to detect and quantify eukaryotic and prokaryotic cells. Because of its simplicity and reliability, BP can be a new tool for quantification of specifically labeled target microorganisms, both in industrial products and natural samples.     

Factors affecting soil-based natural regeneration of <Emphasis Type="Italic">Abies sachalinensis</Emphasis> following timber harvesting in a sub-boreal forest in Japan

Natural regeneration of Abies sachalinensis on soil was studied in a natural sub-boreal forest managed by single tree selection cutting located within the Hokkaido Tokyo University Forest. First, seedlings of A. sachalinensis on skid trails, soil mounds, soil pits, and undisturbed soil were counted, and their areas were measured. Most seedlings were found on skid trails. Seedling densities of A. sachalinensis on sites with soil disturbance, with the exception of soil pits, were significantly greater than that on undisturbed soil. Second, densities of Sasa senanensis, heights of S. senanensis, and seedling densities of A. sachalinensis on skid trails and undisturbed soil in both a closed-canopy stand and in a canopy gap were compared. Seedling density of A. sachalinensis on skid trails in the closed-canopy stand was significantly greater than that in the canopy gap. Average density and average height of S. senanensis, which is known to interfere with regeneration of many woody species, were significantly greater on skid trails and undisturbed soil in the canopy gap than those in the closed-canopy stand. It could be concluded from this study that both crown closure and soil disturbance are essential for natural regeneration of A. sachalinensis on soil in sub-boreal forests.     

Evaluation of nutritional values of selected commercial fish meal sources in turbot (Psetta maxima) diets

This study was conducted to evaluate nutritional values of selected commercial fish meals (FM) with two experiments in diets of turbot. In experiment I, five diets based on two Peruvian anchovy meals (one is a flame‐dried FM: Peru‐1 and the other steam‐dried FM: Peru‐2), a Danish FM (Danish LT), a domestic anchovy LT meal (Domestic LT) and an Alaskan white FM (WFM) as major protein source were formulated to contain 56% protein. The diets were fed to fish with an initial average weight of 119.4 ± 0.1 g for 9 weeks. Growth rate, feed utilization, whole body and visceral compositions, nitrogen, lipid and energy balances did not significantly change in Experiment 1 among the treatments. In Experiment 2, four diets based on three FMs (Domestic LT, Danish LT and Peru‐1) and their mixture (Mix FM) were formulated to contain 50% protein and fed to triplicated groups of fish with an average initial weight of 51.4 ± 0.1 g for 6 weeks. Once again, there were no significant differences in growth rate and feed consumption among the treatments. However, fish on Mix FM diet had significantly better FCR and PER than the others. The results of two experiment indicated that Danish LT and Domestic LT were better in terms of phosphorus (P) retention and losses than two Peruvian FMs and WFM, suggesting that although different sources of FMs were comparable in terms of growth and feed utilization, they could lead to substantial differences in environmental impacts.     

线粒体DNA由来的RAPD标记能鉴别水稻雄性不育和可育细胞质

以野败型细胞质的水稻雄性不育系珍汕97A及其保持系珍汕97B的总DNA为模板,从100个引物中筛选到OPA12对珍汕97A能扩增出一条1600 bp的特异带.用OPA12扩增野败型细胞质的龙特浦A及其保持系龙特浦B、矮败型细胞质的协青早A及其保持系协青早B、恢复系明恢63、珍汕97A/明恢63的F1和F2个体的总DNA,不育系、F1和F2所有调查个体都有16     

Mechanism of reduction of exchangeable aluminum by gypsum application in acid andosols

Mechanism of reduction of exchangeable aluminum in acid Andosols treated with gypsum was studied by using cation exchange resin methods to determine the amount of polymerized aluminum. Two types of acid Andosols were used as test soils: Kitakami light colored Andosol (fine, mixed, mesic, Andic Dystrochrept) and Kawatabi thick high humic Andosol (medial, mesic mixed Alic Pachic Melanudand). Polymerization of aluminum in the soil solution of both Kitakami and Kawatabi Andosols treated with gypsum was suggested based on an analysis using cation exchange resin methods, whereas that in monomer aluminum solution was not detected. Accumulation of polymerized aluminum in both Kitakami and Kawatabi Andosols was determined by using cation exchange resin, and the amounts of polymer aluminum trapped by the resin and the ratio of polymer aluminum to monomer aluminum were increased with the incubation time. The values of CEC which decreased in the Kitakami Andosol after gypsum treatment were almost equivalent to the amounts of cation exchange sites occupied by polymer aluminum ions which were calculated based on the decrease of the values of Y _l. We conclude that the mechanism of reduction of exchangeable aluminum in strongly acid Andosols treated with gypsum is as follows: firstly, exchangeable aluminum adsorbed on the cation exchange sites of soils may be released into the soil solution due to the increase in the ion strength caused by gypsum application, and then monomer aluminum in soil solution may be polymerized in the presence of soil colloidal materials. Consequently, the polymer aluminum formed in the soil solution may be selectively and irreversibly fixed on the cation exchange sites of 2 : 1 clay minerals.     

Richness of Colchic vegetation: comparison between refugia of south-western and East Asia

Background  

Heavy metals, especially copper, nickel, lead and zinc, have adverse effects on terrestrial and in aquatic environments. However, their impact can vary depending on the nature of organisms. Taking into account the ability of heavy metals to accumulate in sediments, extended knowledge of their effects on aquatic biota is needed. In this context the use of model organisms (often unicellular), which allows for rapid assessment of pollutants in freshwater, can be of advantage. Pentachlorophenol has been extensively used for decades as a bleaching agent by pulp- and paper industry. Pentachlorophenol tends to accumulate in the nature. We aim to determine if photosynthesis and motility can be used as sensitive physiological parameters in toxicological studies of Chlamydomonas reinhardtii, a motile green unicellular alga. It is discussed if photosynthesis and motility can be used as sensitive physiological parameters in toxicological studies.     

Kinetic study of the quenching reaction of singlet oxygen by Pyrroloquinolinequinol (PQQH(2), a reduced form of Pyrroloquinolinequinone) in micellar solution

A kinetic study of the quenching reaction of singlet oxygen ((1)O(2)) with pyrroloquinolinequinol (PQQH(2), a reduced form of pyrroloquinolinequinone (PQQ)), PQQNa(2) (disodium salt of PQQ), and seven kinds of natural antioxidants (vitamin C (Vit C), uric acid (UA), epicatechin (EC), epigallocatechin (EGC), α-tocopherol (α-Toc), ubiquinol-10 (UQ(10)H(2)), and β-carotene (β-Car)) has been performed. The second-order rate constants k(Q) (k(Q) = k(q) + k(r), physical quenching and chemical reaction) for the reaction of (1)O(2) with PQQH(2), PQQNa(2), and seven kinds of antioxidants were measured in 5.0 wt % Triton X-100 micellar solution (pH 7.4), using UV-visible spectrophotometry. The k(Q) values decreased in the order of β-Car > PQQH(2) > α-Toc > UA > UQ(10)H(2) > Vit C ～ EGC > EC ? PQQNa(2). PQQH(2) is a water-soluble antioxidant. The singlet oxygen-quenching activity of PQQH(2) was found to be 6.3, 2.2, 6.1, and 22 times as large as the corresponding those of water-soluble antioxidants (Vit C, UA, EGC, and EC). Further, the activity of PQQH(2) was found to be 2.2 and 3.1 times as large as the corresponding activity of lipid-soluble antioxidants (α-Toc and UQ(10)H(2)). On the other hand, the activity of PQQH(2) is 6.4 times as small as that of β-Car. It was observed that the chemical reaction (k(r)) is almost negligible in the quenching reaction of (1)O(2) by PQQH(2). The result suggests that PQQH(2) may contribute to the protection of oxidative damage in biological systems, by quenching (1)O(2).     

Staphylococcus pseudintermedius exfoliative toxin EXI selectively digests canine desmoglein 1 and causes subcorneal clefts in canine epidermis

Staphylococcal exfoliative toxins are known to digest desmoglein (Dsg) 1, a desmosomal cell–cell adhesion molecule, thus causing intraepidermal splitting in human bullous impetigo, staphylococcal scalded skin syndrome and swine exudative epidermitis. Recently, a novel exfoliative toxin gene (exi), whose sequence shares significant homology with previously identified exfoliative toxins, was isolated from Staphylococcus pseudintermedius. Little is known about the pathogenic involvement of this toxin in canine pustular diseases such as impetigo. The aim of this study was to determine whether EXI, the product of the exi gene, digests canine Dsg1 and causes intraepidermal splitting in canine skin. An exi gene was isolated from chromosomal DNA of an S. pseudintermedius strain obtained from a pustule of a dog with impetigo, and was used to produce a recombinant EXI by Escherichia coli expression. When purified recombinant EXI was injected intradermally into normal dogs, it caused the development of vesicles or erosions with superficial epidermal splitting. In addition, the EXI abolished immunofluorescence for Dsg1, but not for Dsg3, at the injection sites. Moreover, the EXI directly degraded baculovirus‐secreted recombinant extracellular domains of canine Dsg1, but not that of canine Dsg3, in vitro. The EXI also degraded mouse Dsg1α and swine Dsg1, but not human Dsg1, mouse Dsg1β and Dsg1γ. Conversely, recombinant SIET, previously designated as S. intermedius exfoliative toxin, did not cause intraepidermal splitting or degradation of any Dsgs. These findings indicate that EXI has a proteolytic activity that digests canine Dsg1, and this characteristic might be involved in the pathogenesis of intraepidermal splitting in canine impetigo.     

Genotyping of Malassezia pachydermatis isolates from canine healthy skin and atopic dermatitis by internal spacer 1 (IGS1) region analysis

Isolates of Malassezia pachydermatis from healthy dog skin and from dogs with atopic dermatitis were molecularly characterized using internal spacer 1 (IGS1) region analyses, and their phospholipase A2 activity and pH growth profiles were then characterized in vitro. The percentage of isolates from healthy dogs that had the following IGS1 subtypes (isotype, %) were as follows: 1A, 6%; 1B, 27%; 1C, 11%; 2A, 6%; 2B, 6%; 3A, 11%; 3C, 3%; and 3D, 24%. In contrast, 9% of isolates from dogs with atopic dermatitis were isotype IB and 91% were isotype 3D, indicating that isolates of subtype 3D were the most prevalent in dogs with atopic dermatitis. Production of phospholipase A2 was statistically higher in isolates of subtype 3D than in the other subtypes. The subtype 3D isolates showed enhanced growth on alkaline medium compared with non-3D subtype isolates. The main clinical sign of canine Malassezia dermatitis is waxy exudates on the skin, which predispose the patient to development of a yeast overgrowth of the subtype 3D. Increased phospholipase A2 production may be involved in the inflammatory process associated with Malassezia dermatitis.     

An azole-resistant isolate of Malassezia pachydermatis

Canine Malassezia dermatitis (MD) is frequently treated with systemic ketoconazole (KTZ) and itaconazole (ITZ). However, the antifungal susceptibility of clinical isolates of M. pachydermatis from dogs and cats to the azoles has not been well investigated. In the present study, the in vitro susceptibility of the standard strain (CBS1879: the neotype strain of M. pachydermatis) and 29 clinical isolates of M. pachydermatis to the azoles was measured by a modified CLSI M27-A2 test using modified Dixon medium as well as by the E-test. The minimum inhibitory concentrations (MICs) of the 30 isolates of M. pachydermatis (including the neotype strain) against KTZ and ITZ were <0.03 μg/ml by the two methods. The MICs of 1 clinical isolate (ASC-11) were 1 and 2 μg/ml against KTZ, and 2 and 8 μg/ml against ITZ, by the modified CLSI M27-A2 test and the E-test, respectively. Thus, isolate ASC-11 may be resistant to these azoles, making this the first report of a resistant isolate of M. pachydermatis to KTZ and ITZ.