Soil seed banks in a mature Hinoki (<Emphasis Type="Italic">Chamaecyparis obtusa</Emphasis> Endl.) plantation and initial process of secondary succession after clearcutting in southwestern Japan

To profile secondary succession after clearcutting a conifer plantation, we examined the composition of the soil seed bank and the process of vegetation recovery after clearcutting a mature Hinoki (Chamaecyparis obtusa) plantation in southwest Japan. We set up two study plots and collected 25 and 26 soil samples (each 20 cm × 30 cm × 5 cm depth), respectively, in April 1999, before clearcutting. Using the seedling emergence method, we detected 500–839 buried viable seeds m⁻² in 42–44 species. Soil seed banks in both plots contained typical pioneer species but few late-successional species. After clearfelling the plantation in June 1999, we censused seedling establishment for 4 years and the composition of the vegetation for 6 years. More than half of the seedlings emerged in the second year, suggesting that the timing of clearcutting is possibly critical to seedling establishment. Nineteen plant species disappeared after clearcutting, 40 species were retained and 43 species newly emerged after clearcutting. Most of the newly emerged species were pioneer species which originated in the soil seed bank. Seven of the pioneer species, as typified by Rubus crataegifolius, showed clonal growth from the third year after clearcutting. Japanese serow browsed the seedlings of major pioneer species, possibly affecting the process of secondary succession. Late-successional species such as Abies firma declined in coverage or disappeared after clearcutting. Our results suggest that the soil seed bank makes a major contribution to vegetation recovery after clearcutting, but possibly not to restoration of the late-successional species.     

Identification and pathogenicity of Pythium species causing damping-off of kidney bean

Five Pythium species (Pythium irregulare, P. mamillatum, P. myriotylum, P. spinosum and P. ultimum var. ultimum) were isolated from the hypocotyls and roots of kidney bean plants with damping-off from a commercial field and from experimental plots that have undergone either continuous cropping with kidney bean or rotational cropping with arable crops. In inoculation tests, all five Pythium species were pathogenic to kidney bean. This is the first report of damping-off of kidney bean caused by Pythium species; we named this disease damping-off of kidney bean. The nucleotide sequence data reported are available in the DDBJ/EMBL/GenBank databases under accession numbers AB291811, AB291944 and AB291945.     

N605ab,a specific molecular marker for <Emphasis Type="Italic">Phytophthora infestans</Emphasis>, distinguishes three genotypes in Japan

Random amplified polymorphic DNA (RAPD) analysis using the OPG-06 primer generated specific patterns for Japanese genotypes US-1, JP-1, and a new A1 (JP-2, JP-3, and JP-4) of Phytophthora infestans. N605, a specific RAPD fragment, was cloned and sequenced. PCR primers BD1/BD2 were constructed based on the N605 sequence and were used to clarify the genotypes. PCR products using the BD1/BD2 primers (N605ab marker) easily distinguished the new A1 from US-1 and JP-1. This technique provides a simple and effective method for rapid genotype discrimination that can be used in ecological experiments and forecasts for the occurrence of late blight.     

Isolation of antifungal-resistant yeasts from bulk milk in Japan

We isolated ascomycetous yeasts including Candida species, that originally belonged to the genus Candida, from bulk milk in the Aichi area of Japan, and determined the minimum inhibitory concentrations (MICs) of antifungal drugs on these isolates by conducting E-tests. We isolated 7 human pathogenic species (14 isolates) from 14 bulk milk samples: 5 Candida species of yeasts, and 2 Candida-related species. Two isolates of C. albicans and C. inconspicua were resistant to fluconazole (MIC >32 mg/l). One isolate of C. krusei was resistant to both azoles (fluconazole: >256 mg/ml and itraconazole: 4 mg/l). One isolate of C. catenulata might be resistant to amphotericin B (>32 mg/l).     

Identification of vitamin B12 and pseudovitamin B12 from various edible shellfish using liquid chromatography–electrospray ionization/tandem mass spectrometry

In this study, the vitamin B₁₂ contents were analyzed in the edible portions of various shellfish (bivalves and snails). High vitamin B₁₂ contents (30.5–53.3 μg/100 g wet weight) were detected in mussels, surf clams, bloody clams, and freshwater clams. However, scallops and abalone had extremely low vitamin B₁₂ contents (0.1–1.1 μg/100 g wet weight) which was attributed to only the muscle portions being edible. These results suggest that high levels of vitamin B₁₂ are accumulated in the viscera of shellfish. Vitamin B₁₂ levels were also significantly higher in bivalves than in snails. The corrinoid compounds purified from all bivalves were identified as “true” vitamin B₁₂ using liquid chromatography–electrospray ionization/tandem mass spectrometry. In edible snails, abalone, and pond snails, however, both vitamin B₁₂ and pseudovitamin B₁₂ (an inactive corrinoid) were observed to be the major and minor corrinoid compounds, respectively. Based on these results, we conclude that the whole bodies of these edible bivalves are excellent sources of vitamin B₁₂ for humans.     

Molecular mechanism of fertilization in the pig

At fertilization, the sperm triggers resumption from the arrest, extrusion of the second polar body and pronuclear formation, the events of which are collectively acknowledged as ‘oocyte activation’. In all species up to date, oocyte activation requires a fertilization‐associated increase in the intracellular concentration of calcium. Especially in mammals, the signal of intracellular calcium rise at fertilization consists of periodical rises, which are also referred to as calcium oscillations. Our recent results suggest that these calcium oscillations have an important role in not only oocyte activation but also development of mammals. Pigs are animals of great agricultural value and ones in which assisted reproductive techniques, including somatic cell nuclear transfer, to produce gene‐modified pigs. Although reconstructed embryos require artificial activation stimuli which mimic fertilization‐associated increase of intracellular calcium in the oocytes, it has been known that the developmental ability of the oocytes after artificial activation is low and the regimen seems to be required for improvement. Recently we focused on two molecules, phospholipase C zeta and inositol 1,4,5‐triphosphate receptor which have important roles in regulation of calcium oscillations during fertilization in mammals, including pigs. In this review, we will discuss the present status and future perspective of molecular mechanisms during fertilization in pigs.     

Lignosulfonate/APP IFR and its flame retardancy in lignosulfonate-based rigid polyurethane foams

Lignin containing substantial aromatic rings and high content of carbon has been employed as carbonizing agent to investigate the flame retardancy in the lignin/ammonium polyphosphate (APP) intumescent flame retardant (IFR) system. In addition, owing to the abundant phenolic and aliphatic hydroxyl groups, lignosulfonate, which is considered as a renewable aromatic macropolyols, substituted part of diethylene glycol (DEG) and copolymerized with isocyanate to produce lignosulfonate-based rigid polyurethane (LRPU) foams. Thermal stability was characterized by thermogravimetric analysis (TGA), and flame retardancy was investigated by limiting oxygen index (LOI) and cone calorimetry testing (CCT). Lignosulfonate increases thermal stability of LRPU foams and LRPU containing 15 wt% of lignosulfonate based on DEG (L₁₅RPU) give rise to the best thermal stability. When 15% of lignosulfonate incorporated in the LRPU, reduced the heat release rate (HRR) and total heat release (THR) value 21 kW/m² and 13 MJ/m², respectively, and postponed 96 s time-to-peak carbon monoxide production than that of pure DEG rigid polyurethane (RPU) foam, the LOI values increased progressively with lignosulfonate content increasing. These results showed that lignosulfonate polyol may substitute polyol to produce lignosulfonate-based RPU foam and the presence of lignosulfonate could improve the flame retardancy. The mass loss gradually decreases with increasing APP addition, and the highest char yield was obtained from LRPU5 foam which at the lignosulfonate-to-APP ratio is 1:5. At the lignosulfonate-to-APP ratio of 1:5, the LOI value increased over 30%, and the HRR value reduced and the time-to-peak HRR postponed significantly. In addition, LRPU5 foams give rise to the lowest effect heat combustion (EHC) value, less smoke, and carbon monoxide (CO) production. Lignosulfonate acts as carbonizing agent in the lignosulfonate/APP IFR system, and the best fire retardancy is obtained at 1:5 of lignosulfonate-to–APP ratio.     

Flow cytometric analysis of peripheral blood mononuclear cells induced by experimental endotoxemia in horse

Cellular activation and functional cell surface markers were evaluated during experimentally-induced endotoxemia in healthy horses. Eight healthy adult horses were infused a low dose of endotoxin (lipopolysaccharide from Escherichia coli O26: B6, 30 ng/kg of body weight, IV) and five control horses were given an equivalent volume of sterile saline solution. Venous blood samples were collected for flow cytometric analysis of peripheral blood mononuclear cells (PBMCs) and to measure plasma endotoxin concentrations. Clinical signs of endotoxemia were recorded at 10, 20, 30, 40, 50 min, 1, 2, 3, 4, 8, 16, 24 and 48 hr after endotoxin or saline solution administration. Clinical findings characteristic of endotoxemia (tachycardia, tachypnea, increased rectal temperature, and leukopenia) occurred transiently in all horses administered endotoxin; however, plasma endotoxin concentrations were detectable in only 50% (4/8) of the endotoxin-infused horses. The percentage of CD4(+), CD5(+), and CD8(+) cells decreased while the percentage of CD14(+), IgM(+), and MHC class II(+) cells increased significantly after endotoxin infusion. Alterations in the immunophenotype of PBMCs from horses with experimentally-induced endotoxemia were associated with changes in vital signs, indicating that endotoxin altered the immuno balance.