Cellular injury and lipid peroxidation induced by hexavalent chromium in isolated rat hepatocytes

In order to elucidate the relationship between cellular injury and lipid peroxidation induced by hexavalent chromium (CrVI), isolated rat hepatocytes treated with any one of scavengers of active oxygen species, antioxidants or antichromium agent were incubated with K2Cr2O7 as CrVI (1 mM Cr). After the incubation, the development of lipid peroxidation was determined as thiobarbituric acid (TBA)-reacting materials in total lipid extracts from the incubated hepatocytes. Cellular injury was observed as a leakage of lactate dehydrogenase (LDH) from hepatocytes into incubation medium. The contents of reduced glutathione (GSH) in hepatocytes were also assessed. Results obtained were as follows: (1) CrVI facilitated lipid peroxidation in isolated hepatocytes after 20 min of incubation. On the other hand, the cellular injury induced by CrVI was barely observed even after 60 min of incubation. (2) The CrVI-induced lipid peroxidation was inhibited by catalase and mannitol as scavengers of active oxygen species, or N,N'-diphenyl-p-phenylenediamine and alpha-tocopherol as antioxidants. However the cytotoxicity of CrVI could not be prevented by these chemicals. (3) CrVI depleted the contents of intracellular GSH and diminished the activities of glutathione reductase (GR) and glutathione-S-transferase (GST) except glutathione peroxidase. (4) The scavengers of active oxygen species and the antioxidants could not prevent the depletion of intracellular GSH induced by CrVI. (6) Ascorbic acid, antichromium agent, prevented all of the lipid peroxidation, the cellular injury and intracellular GSH depletion induced by CrVI.(ABSTRACT TRUNCATED AT 250 WORDS)     

Changes in the expression of toll-like receptor mRNAs during follicular growth and in response to lipopolysaccharide in the ovarian follicles of laying hens

The aim of this study was to determine the changes in the mRNA expression of Toll-like receptors (TLRs) in hen ovarian follicles during follicular growth and in response to lipopolysaccharide (LPS). White follicles and the fifth largest to largest follicles (WF and F(5)-F(1), respectively) were collected from laying hens. To examine the effects of LPS, the laying hens were treated intravenously with LPS (1 mg/kg BW) 0, 3, 6, 12 and 24 h before examination. Expressions of TLRs and IL-1beta in the theca and granulosa layers were examined by semi-quantitative RT-PCR. Immunocytochemistry was performed to identify immunoreactive TLR-4. The theca layer expressed TLR-2, TLR-4, TLR-5 and TLR-7, whereas the granulosa layer expressed only TLR-4 and TLR-5. The expression of TLR-4 and TLR-5 in the theca layer increased significantly during follicular growth. In the granulosa layer, the expression of TLR-5 increased, but that of TLR-4 was unchanged. Expression of TLR-4 increased significantly during the period of 6 to 12 h of LPS treatment in the theca layer and during the period of 12 to 24 h in the granulosa layer of F(3). Immunoreaction products for TLR-4 were observed in theca interna and granulosa layers of WF and F(5)-F(1), with the greater amount observed in the theca interna. LPS treatment significantly increased expression of IL-1beta in the theca layer after 3 h and in the granulosa layer during the period of 12 to 24 h. These results suggest that TLRs are expressed in ovarian follicles and that TLR-4 and TLR-5 expression increase with the growth of follicles. Enhanced expression of TLR-4 and IL-1beta by LPS in the theca and granulosa layers suggests possible roles of TLR in recognition of microorganisms.     

Pine Wilt Disease Caused by the Pine Wood Nematode: The Induced Resistance of Pine Trees by the Avirulent Isolates of Nematode

Since the beginning of the 20th century, pine trees in Japan have been seriously damaged by the pine wilt disease. This disease is caused by the pine wood nematode, Bursaphelenchus xylophilus, which is transmitted by the Japanese pine sawyer, Monochamus alternatus. The control of disease depends to a large extent on chemicals, but the public is now demanding environmentally friendly control methods. The virulence of B. xylophilus varies very widely. Pre-inoculation of young pine trees in a nursery with avirulent B. xylophilus has induced systemic resistance of trees against a subsequent inoculation with virulent B. xylophilus. This induced resistance was considered a hopeful means for developing a biological control for the disease. The induced resistance by the avirulent nematodes was also expressed in mature pine trees in a forest where the disease was naturally epidemic. However, the effects of induced resistance were not satisfactory for practical biological control. Since the inoculation with higher concentrations of the avirulent B. xylophilus induced the resistance more effectively, the pre-inoculation method will need to be improved to develop the biological control. The induced resistance of pine trees by avirulent B. xylophilus should be one of the candidate biological control methods against pine wilt disease. This induced resistance also provides an experimental system to clarify physiological interactions between the nematodes and pine trees.     

Increased plasma levels of soluble intercellular adhesion molecule-1 (sICAM-1) and soluble vascular cell molecule-1 (sVCAM-1) associated with disease severity in a primate model for severe human malaria: Plasmodium coatneyi-Infected Japanese macaques (Macaca fuscata)

In the present study, we investigated plasma levels of soluble intercellular adhesion molecule-1 (sICAM-1) and vascular cell adhesion molecule-1 (sVCAM-1) in seven Japanese macaques (Macaca fuscata) infected with Plasmodium coatneyi. Concentrations of sICAM-1 and sVCAM-1 were significantly elevated in the severe phase; the levels were maximally increased up to six times and three times those before infection, respectively. We subsequently examined kinetic profiles of sICAM-1 and sVCAM-1 concentration in plasma obtained from two infected monkeys. Both infected monkeys had markedly increased levels of these adhesion molecules when they exhibited severe clinical signs correlated with rapid increase in parasitemia. These results suggest that the elevation of levels of sICAM-1 and sVCAM-1 is a critical step in the pathogenesis of severe malaria in vivo.     

Gene constitution of South-East Asian native chickens, commercial chickens and jungle fowl using polymorphisms of four calpain genes

The gene constitution of polymorphisms of the four calpain genes (µ‐calpain, m‐calpain, p94, and µ/m‐calpain) were analyzed in South‐East Asian native chickens, White Leghorn and Broiler commercial chickens, and Red and Green jungle fowl. Polymorphisms were detected at all loci in chickens and Red jungle fowl, but only for CAPN1 (µ‐calpain gene) in Green jungle fowl. CAPN2 and CAPN1.5 are linked on chicken chromosome 3, and the genotype for these loci were treated as haplotype. Some combinations of calpain loci were tested using principal component analysis, and the best combination (CAPN1, CAPN3, and CAPN1.5) was determined. The proportion of polymorphic loci (P_poly) and heterozygosity (H?) were 1.00 and 0.316–0.465 in domestic chickens and red jungle fowl, and 0.33 and 0.137 in Green jungle fowl, respectively. G_ST values suggested that the degree of subdivision among native chickens was relatively low except for Thailand, which was highest. Pair‐wise F_ST testing, dendrogram and principal component analysis from the results of calpain loci showed that the four South‐East Asian native and commercial chicken populations were close genetically.     

Leaf-fall phenology along a topography-mediated environmental gradient in a cool–temperate deciduous broad-leaved forest in Japan

Leaf-fall phenology was studied in a cool–temperate deciduous broad-leaved forest in central Japan in relation to the topographic environmental gradients that occur along a short mountain slope. Leaf-fall phenology was described quantitatively using data from leaf litter collected along the slope. In autumn, leaf fall at the study site tended to occur slightly earlier on the upper slope than on the lower slope. This pattern was found at both the stand and the species levels. Our results suggest that leaf-fall phenology may be affected by difference in microclimatic conditions, because environmental conditions are thought to be more severe on the upper slope than on the lower slope. The less intensive methods used in this study, the litter trap method, and Dixon’s model succeeded in quantifying the phenological patterns of leaf fall within stands and within species along the short mountain slope.     

Ectomycorrhizal fungal communities of Dipterocarpus alatus seedlings introduced by soil inocula from a natural forest and a plantation

We attempted to introduce ectomycorrhizal (ECM) fungi onto seedlings of Dipterocarpus alatus (Dipterocarpaceae) by soil inocula collected from tree stands of D. alatus. Top soils collected from beneath the trees of D. alatus in a dry evergreen forest and a 15-year-old plantation were inoculated onto germinating seeds of D. alatus. After 7 months of seedling cultivation, ECM fungal communities in the seedlings were investigated based on the sequences of ITS rDNA. The ECM fungi detected were divided into 19 phylotypes by molecular analysis. Most of the phylotypes were identified as ECM fungal taxa, i.e., Clavulina, Laccaria, Lactarius, Tomentella, Pyronemataceae, and Tricholomataceae. Accordingly, we can confirm that soil inoculation is a simple method to induce ECM formation with diverse fungi in pot cultured seedlings, which would be useful for introducing diverse ECM fungi to dipterocarp plantations.     

Mercury content and its bioconcentration factors in wild mushrooms at Łukta and Morag,northeastern Poland

Total concentrations of mercury were determined using cold-vapor atomic absorption spectroscopy (CV-AAS) in the fruiting bodies of 16 species of wild mushrooms and underlying soil (0-10 cm) substrates collected in the areas of the Communes of Morag and ?ukta in the county of Ostróda in northeastern Poland in 1997-1998. A total of 174 composite samples of caps, 174 stalks, 80 whole fruiting bodies (collectively 1254 specimens), and 252 soils were examined. Among several species of mushrooms analyzed, the greatest concentrations were between 1300 and 71000 ng.g(-1) of dry matter. These levels were found in the caps of Sweating mushroom (Clitocybe rivulosa), King Bolete (Boletus edulis), and Common Puffball (Lycoperdon perlatum) and also were characterized by the highest bioconcentration factors (BCF) for Hg, which ranged between 160 +/- 82 and 110 +/- 34. The cap to stalk quotient for mercury concentrations was approximately 2 for most of the species except Poison Pax (Paxilus involutus), which had a greater concentration in caps than in stalks and a quotient of 4.4 +/- 7.2. Hg concentrations in the underlying soil substrates (0-10 cm layer) ranged between 21 +/- 21 and 390 +/- 130 ng.g(-1) of dry matter. The results showed that the consumption of mushrooms, considered to be the sole dietary source of mercury at the highest or mean element concentrations found, is not hazardous at daily ingestion rates of less than 70 and 210 g of fresh product, which would result in a hazard index value of less than unity.