Allelic variation of a clubroot resistance gene (Crr1a) in Japanese cultivars of Chinese cabbage (Brassica rapa L.)

Clubroot resistance (CR) is an important trait in Chinese cabbage breeding worldwide. Although Crr1a, the gene responsible for clubroot-resistance, has been cloned and shown to encode the NLR protein, its allelic variation and molecular function remain unknown. Here, we investigated the sequence variation and function of three Crr1a alleles cloned from six CR F₁ cultivars of Chinese cabbage. Gain-of-function analysis revealed that Crr1a^Kinami90_a isolated from the cv. ‘Kinami 90’ conferred clubroot resistance as observed for Crr1a^G004. Because two susceptible alleles commonly lacked 172 amino acids in the C-terminal region, we investigated clubroot resistance in transgenic Arabidopsis harboring the chimeric Crr1a, in which 172 amino acids of the functional alleles were fused to the susceptible alleles. The fusion of the C-terminal region to the susceptible alleles restored resistance, indicating that their susceptibility was caused by the lack of the C-terminus. We developed DNA markers to detect the two functional Crr1a alleles, and demonstrated that the functional Crr1a alleles were frequently found in European fodder turnips, whereas they were rarely introduced into Japanese CR cultivars of Chinese cabbage. These results would contribute to CR breeding via marker-assisted selection and help our understanding of the molecular mechanisms underlying clubroot resistance.     

Molecular dynamics of heterochromatin protein 1beta, HP1beta, during mouse preimplantation development

To elucidate the molecular dynamics of HP1beta in mouse preimplantation embryos, we examined the localization, dynamics, and mobility of HP1beta in the (pro)nucleus by live cell imaging. Time-lapse observation revealed that the chromatin association of HP1beta is regulated in a cell cycle-dependent manner. HP1beta was localized in the interphase nucleus and was dynamically dissociated from the nucleus during the metaphase stage. The HP1beta assembly and clustered heterochromatin structure were both found in the nuclei of 2-cell and later-stage embryos. Moreover, fluorescent recovery after photobleaching analysis implied that HP1beta is more freely mobile in the pronucleus of the 1-cell embryo than in the 4-cell nucleus. These results suggest that the chromatin configuration may be regulated by the stability and mobility of chromatin-associated proteins including HP1beta during early embryonic stages.     

A new method for evaluating symplastic cadmium absorption in the roots of Solanum melongena using enriched isotopes 113Cd and 114Cd

Radioisotope techniques are well known as methods for evaluating symplastic ion absorption in roots. In the present study, a new method for evaluating symplastic cadmium (Cd) absorption in plant roots was developed using the enriched isotopes ¹¹³Cd and ¹¹⁴Cd. Seedlings of Solanum melongena were exposed to an enriched isotope solution of ¹¹³Cd at 25°C for 30 min. The roots were excised from each seedling and were then immersed in a cold buffer solution without Cd at 2°C for 120 min to suppress the metabolic activity of the roots. Finally, the roots were treated with a cold buffer solution containing enriched stable isotope ¹¹⁴Cd at 2°C for 120 min, whereby the apoplastically bound ¹¹³Cd was desorbed. We tested the validity of our method for evaluating symplastic Cd in roots compared with the conventional method based on differences in the amount of Cd absorbed at 2°C and 25°C using unlabeled Cd. There was no difference in the symplastic Cd content of the roots between the two methods. These results indicate that it is possible to evaluate the symplastic Cd content in roots using the enriched isotopes ¹¹³Cd and ¹¹⁴Cd.     

Study of the Structure–Activity Relationship of an Anti-Dormant Mycobacterial Substance 3-(Phenethylamino)Demethyl(oxy)aaptamine to Create a Probe Molecule for Detecting Its Target Protein

The current tuberculosis treatment regimen is long and complex, and its failure leads to relapse and emergence of drug resistance. One of the major reasons underlying the extended chemotherapeutic regimen is the ability of Mycobacterium tuberculosis to attain a dormant state. Therefore, the identification of new lead compounds with chemical structures different from those of conventional anti-tuberculosis drugs is essential. The compound 3-(phenethylamino)demethyl(oxy)aaptamine (PDOA, 1), isolated from marine sponge of Aaptos sp., is known as an anti-dormant mycobacterial substance, and has been reported to be effective against the drug resistant strains of M. tuberculosis. However, its target protein still remains unclear. This study aims to clarify the structure–activity relationship of 1 using 15 synthetic analogues, in order to prepare a probe molecule for detecting the target protein of 1. We succeeded in creating the compound 15 with a photoaffinity group that retained antimicrobial activity, which proved to be a suitable probe molecule for identifying the target protein of 1.     

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.     

Seasonal changes in serum vitamin A, vitamin E and beta-carotene concentrations in Japanese Black breeding cattle in Hyogo prefecture

The influence of the season on serum vitamin A, E and beta-carotene concentrations was studied in Japanese Black breeding cattle. Blood samples were collected from 13 cows once a month for a year to determine the serum vitamin concentrations. Mean serum concentrations (+/- SE) of retinol, alpha-tocopherol and beta-carotene were 21.3 +/- 0.5 microg/dl (range 9.9 to 44.9), 215.9 +/- 8.0 microg/dl (range 59.7 to 551.6) and 67.3 +/- 5.1 microg/dl (range 10.6 to 425.2), respectively. All these concentrations were lower than the recommended levels. Serum retinol and alpha-tocopherol concentrations in summer were lower than those in winter. These results suggest that the vitamin A, E and beta-carotene status of Japanese Black breeding cows is inadequate, and feeding high quality forages or dietary vitamin supplementation during summer should be recommended.     

Lens formation in the absence of optic cup in rat embryos irradiated with soft X-ray

In order to investigate the effect of soft X-ray irradiation on ocular development, pregnant rats were exposed to a single 12.5 Gy irradiation on embryonic day 9 (ED 9). The embryos obtained by laparotomy on ED 12 and 21 were examined for ocular abnormalities under a binocular stereo-microscope and a light microscope. The ED 12 embryos were stained with osmium tetroxide to facilitate the observation. The stereo-microscopic examination on ED 12 and 21 revealed various types of ocular abnormalities characterized primarily by aplasia or hypoplasia of the optic cup and invaginated lens placode. The light microscopic examination further confirmed these findings histomorphologically, and the hypoplastic abnormalities were classified into three types: (1) hypoplasia of the optic cup and invaginated lens placode, (2) complete malformation of the optic cup and hypoplasia of the invaginated lens placode, and (3) complete malformation of the optic cup and invaginated lens placode. Because the lens was formed in the complete absence of the retina, the development and differentiation of the retina and lens do not seem to be tightly synchronized. Thus, this sequential analysis on ocular abnormalities during the early stage of development supports the notion that the presence of the retina is not always necessary for the development of the lens.     

Isolation and species distribution of staphylococci from animal and human skin

From April 1999 to December 2000, a survey was made on the distribution of Staphylococcus species on the skin of 7 kinds of animals and humans. Staphylococci were isolated from 12 (100%) of 12 pigs, 17 (89.5%) of 19 horses, 30 (100%) of 30 cows, 73 (90.1%) of 81 chickens, 10 (40%) of 25 dogs, 23 (76.7%) of 30 laboratory mice, 20 (52.6%) of 38 pigeons, and 80 (88.9%) of 90 human beings. The predominant staphylococci isolated from a variety of animal species were novobiocin-resistant species, S. xylosus and S. sciuri regardless of the animal host species. The novobiocin-resistant species including S. xylosus and S. sciuri were only occasionally isolated from human skin. The predominant staphylococci found on human skin were novobiocin-sensitive species, S. epidermidis (63.8%), followed by S. warneri (28.8%) and S. hominis (13.8%). The results suggest that the staphylococcal flora inhabiting animal skin are different from those of human skin in regard to the predominant species isolated. In this study, we used pulsed-field gel electrophoresis to examine the chromosomal polymorphisms of S. epidermidis isolated most frequently from human skin. Strains of S. epidermidis showed the greatest genomic diversity in their fragment patterns.     

Atmospheric lead and cadmium deposition within forests in the Kanto district, Japan

Atmospheric lead and cadmium deposition in bulk precipitation and throughfall was investigated at four forests in the Kanto district, Japan, to assess the impact of human activities on the environmental health of forests. Annual lead and cadmium depositions in bulk precipitation ranged from 8.9 to 25.7 g ha⁻¹ year⁻¹ and from 0.77 to 1.30 g ha⁻¹ year⁻¹, respectively. Lead and cadmium deposition increased in the summer at every forest due to large amounts of rainfall. At one of the forests, the depositions were also high in the winter due to heavy snowfall. These depositions were similar to recent depositions observed at other rural and urban sites in Japan and several forests in Europe and North America after 1990. These results indicate that although anthropogenic lead and cadmium are deposited at these rates over wide areas, depositions are still higher than in remote areas.