Identification of AFLP makers linked to non-seed shattering locus (sht1) in buckwheat and conversion to STS markers for marker-assisted selection.

Shattering habit in buckwheat has two forms: brittle pedicel and weak pedicel. Brittle pedicel is observed in wild buckwheat, but not in cultivated buckwheat. Brittle pedicel in buckwheat is produced by two complementary, dominant genes, Sht1 and Sht2. The sht1 locus is linked to the S locus; almost all common buckwheat cultivars possess the allele sht1. To detect molecular makers linked to the sht1 locus, we used amplified fragment-length polymorphism (AFLP) analysis in combination with bulked segregant analysis of segregating progeny of a cross between a non-brittle common buckwheat and a brittle self-compatible buckwheat line. We screened 312 primer combinations and constructed a linkage map around the sht1 locus by using 102 F2 plants. Five AFLP markers were linked to the sht1 locus. Two of these, e54m58/610 and e55m46/320, cosegregated with the sht1 locus without recombination. The two AFLP markers were converted to STS markers according to the sequence of the AFLPs. The STS markers are useful for marker-assisted selection of non-brittle pedicel plants and provides a stepping-stone for map-based cloning and characterization of the gene encoding non-brittle pedicel.

Susceptibility of T cell receptor-alpha chain knock-out mice to ultraviolet B light and fluorouracil: a novel model for drug-induced cutaneous lupus erythematosus.

The anticancer agent 5-fluorouracil (FU) frequently induces cutaneous lupus erythematosus (LE) lesions on sun exposed sites. Based on this observation, we have tried to establish a cutaneous LE model of C57BL/6 J (B6) mice, B6 T cell receptor (TCR)-alpha(-/-) mice and B6 TCR-delta(-/-) mice treated with FU and/or ultraviolet B light (UVBL) in order to clarify the role of T cells and the cytokine profile of cutaneous lupus lesions. Cutaneous LE-like skin lesions could be induced in TCR-alpha(-/-) mice with low FU (0.2 mg) plus UVBL, and in B6 mice treated with a high dose of FU (2.0 mg) plus UVBL. In contrast, low FU plus UVBL induced such skin lesions in TCR-delta(-/-) mice at a very low incidence. Specifically, the skin lesions of TCR-alpha(-/-) mice with low FU plus UVBL appeared more rapidly and were more severe than lesions in B6 mice. The former had the common characteristic features of human chronic cutaneous LE such as typical histology, positive IgG at the dermoepidermal junction, low antinuclear antibody and low mortality. Furthermore, a Th1 response was induced in the development of drug-induced cutaneous LE. FU and UVBL-induced cutaneous LE-like eruption is an excellent model for better understanding the pathomechanisms of skin lesion development in LE.

Vitamin E reduces glucocorticoid-induced growth inhibition and lipid peroxidation in rats.

This experiment was conducted to study the effects of vitamin E on growth inhibition and lipid peroxidation in rats treated with different levels of corticosterone (CTC). Rats (Sprague-Dawley strain, 5 weeks of age) were divided into two groups: control group receiving a basal diet containing 60 mg DL-alpha-tocopheryl acetate/kg diet, and vitamin E group receiving the same diet supplemented with 5,000 mg tocopherol. After 6 days, rats of both diet groups were further divided into three groups by dose levels of CTC treatment (0, 25, and 100 mg CTC/kg body weight/d). CTC was administered to the rats by subcutaneous injection for 4 d. Growth was dose-dependently inhibited by the CTC treatment. Feeding the vitamin E diet significantly (p < 0.05) improved growth retardation. Feed efficiency was lowered by CTC treatment, while this was significantly (p < 0.05) minimized by feeding the vitamin E diet. Lipid peroxidation (TBARS) in the liver was elevated by the CTC treatment (p < 0.001) when the rats were fed the basal diet. The increment in TBARS was significantly (p < 0.001) reduced by vitamin E. The activities of glutathione S-transferase (GST) and superoxide dismutase (SOD) were significantly reduced by the CTC treatment in a dose-dependent manner in both dietary groups. Feeding vitamin E significantly (p < 0.001) improved the reduction in GST activity. The SOD activity showed some tendency. The present results demonstrate the effectiveness of vitamin E in improving growth retardation in glucocorticoid-treated rats and suggest that reductions in increased lipid peroxidation due to CTC may be an important factor of the action of vitamin E.

Vitamin E reduces glucocorticoid-induced oxidative stress in rat skeletal muscle.

The purpose of this study was to investigate the effect of vitamin E on oxidative stress in the skeletal muscle of glucocorticoid-treated rats. Male Sprague-Dawley rats (5 weeks of age) were fed a basal diet or a diet supplemented with vitamin E (5,000 mg DL-alpha-tocopheryl acetate/kg diet) for 10 d. The rats of both diet groups received subcutaneous injections of corticosterone (CTC) (0, 25, and 100 mg/kg body weight/d) during the final 4 d. Weights of the extensor digitorum longus and gastrocnemius (GAST) muscles were dose-dependently reduced by CTC. However, the muscle weight losses in rats fed the vitamin E diet were smaller than those in rats fed the basal diet. Protein carbonyl content in the GAST muscle, which was determined as an index of oxidatively modified protein, was increased by 100 mg of CTC, and the increment was significantly (p < 0.01) reduced by vitamin E supplement. Hyperglycemia was induced by 100 mg of CTC, but it was not affected by vitamin E. Lipid peroxide (TBARS) in plasma and in GAST muscle was elevated by 100 mg of CTC, and vitamin E significantly (p < 0.001) suppressed the formation of TBARS in the muscle. The change in TBARS paralleled that in protein carbonyl. These results show that CTC leads to oxidative stress in rat skeletal muscles and that vitamin E has roles in reducing the oxidative stress which causes muscle atrophy.

Isolation and functional identification of a novel cDNA for astaxanthin biosynthesis from Haematococcus pluvialis, and astaxanthin synthesis in Escherichia coli.

We succeeded in isolating a novel cDNA involved in astaxanthin biosynthesis from the green alga Haematococcus pluvialis, by an expression cloning method using an Escherichia coli transformant as a host that synthesizes beta-carotene due to the Erwinia uredovora carotenoid biosynthesis genes. The cloned cDNA was shown to encode a novel enzyme, beta-carotene ketolase (beta-carotene oxygenase), which converted beta-carotene to canthaxanthin via echinenone, through chromatographic and spectroscopic analysis of the pigments accumulated in an E. coli transformant. This indicates that the encoded enzyme is responsible for the direct conversion of methylene to keto groups, a mechanism that usually requires two different enzymatic reactions proceeding via a hydroxy intermediate. Northern blot analysis showed that the mRNA was synthesized only in the cyst cells of H. pluvialis. E. coli carrying the H. pluvialis cDNA and the E. uredovora genes required for zeaxanthin biosynthesis was also found to synthesize astaxanthin (3S, 3'S), which was identified after purification by a variety of spectroscopic methods.

Studies on serum tocopherol and selenium levels and blood glutathione peroxidase activities in lambs with white muscle disease.

For the investigation of the cause of white muscle disease (WMD), tocopherol (Toc) and selenium (Se) levels and blood glutathione peroxidase (GSH-Px) activities were examined using lambs with WMD and their ewes. Serum Se levels of 4 lambs with WMD were low under 30 ppb, lambs showing very low levels below 15 ppb. The serum Se level was correlated with blood GSH-Px activity showing remarkably low activities in the lambs with WMD. Se contents in the organs of lambs with WMD were lower than those of control lambs, and particularly liver Se contents were deficient levels below 50 ppb. Serum Toc levels were normal, but alpha-Toc contents in organs showed very low levels, especially in the liver. The serum Toc and Se levels and blood GSH-Px activities of their ewes and other sheep kept in the same farm revealed similar results to those of lambs with WMD. Feedstuffs supplied on the farm showed the deficient level of the Se content below 50 ppb and a very low level of alpha-Toc. It was concluded that WMD of lambs in Hokkaido was nutritional muscular dystrophy resulted from deficiencies of Toc and Se to their ewes.

Endoscopic ultrasonography of lymph nodes surrounding the upper GI tract.

We investigated the usefulness of endoscopic ultrasonography (EUS) of lymph nodes surrounding the upper GI tract and tried the enhanced EUS by the method of the oral administration of '10% oil-in-water-type emulsion.' The results were as follows: The ultrasonographic visualization rate of lymph nodes surrounding the esophagus was 33.7% in total; however, it was 43.4% for those greater than 5 mm and 58.7% for those greater than 10 mm. The frequency of lymph node metastasis of esophageal cancer was 48.1% for those larger than 10mm with a round shape and 14.3% for the same size with an ellipsoid shape; for those less than 10mm, it was also low. Lymph nodes surrounding the esophagus and the stomach were enhanced by administration of 10% oil-in-water-type emulsion. The visualization rate of lymph nodes can be increased by using this new method. Endoscopic ultrasonography is very useful for the detection of swelling lymph nodes surrounding the upper GI tract before the operation.