Search

In this research we eliminated chrysanthemum stunt viroid (CSVd) from a highly infected chrysanthemum cultivar using a newly established method. ‘Piato’ is one of the most difficult cultivars in which to obtain CSVd-free plants by conventional methods. Leaf primordium-free shoot apical meristems (LP-free SAMs) of ‘Piato’ plants were dissected and attached to CSVd-free chrysanthemum or cabbage root tips. As shown by nested-PCR, CSVd was not detected in some shoots regenerated on both types of root tip. The production rates of CSVd-free plants using chrysanthemum and cabbage root tips were 14% and 3%, respectively. Regeneration of plants from LP-free SAMs of chrysanthemum plants by attaching these SAMs to root tips is an efficient method of generating CSVd-free chrysanthemum plants.

A new method to regenerate plants from leaf primordia-free shoot apical meristem domes (LP-free SAMs) was developed by establishing the meristem dome on the cut surface of root tips. Ten days after culture, the viable rate of LP-free SAMs of chrysanthemum ‘Piato’ attached to chrysanthemum root tips was >40%. Shoot regeneration was not observed from LP-free SAMs without the root tips. When LP-free SAMs of chrysanthemum were transferred to root tips of either petunia, cabbage, or carnation, the highest shoot regeneration rate was observed with cabbage root tips. Microscopic observation documented that the LP-free SAM temporarily adhered to the cut surface of the root tip of cabbage.

Pathologic and immunohistochemical changes caused by group I of the fowl adenovirus (FAV) serotype-1 99ZH strain, isolated from broiler chickens exhibiting gizzard erosion, were investigated in commercial broiler chickens. One hundred twenty-two chickens were inoculated with the strain by both oral and ocular routes at 1, 3, or 5 weeks of age and euthanatized for necropsy within 4–18 days of inoculation. Focal gizzard erosions were observed in the inoculated chickens of each age group. A histologically degenerative koilin layer, necrotic mucosa, intranuclear inclusion bodies in the glandular epithelial cells, inflammatory cell infiltrations in the lamina propria, submucosa, and a muscle layer were seen in the gizzards. Immunohistochemical staining showed evidence of FAV antigens in the intranuclear inclusion bodies. These findings were recognized regardless of their maternal antibody levels for FAV serotype-1. Gizzard lesions appeared later in the lower-dose-inoculated chickens than in the higher-dose–inoculated chickens. Numerous CD3-positive cells and IgY-positive plasma cells were seen in the gizzard lesions. In 5-week-old chickens the heterophil infiltrations in the lesions were milder than in younger chickens. Intranuclear inclusion bodies also were observed in the epithelial cells of the ileum or cecal tonsils of some chickens. Thus, this study shows that FAV-99ZH causes adenoviral gizzard erosion in broiler chickens without hepatic or pancreatic lesions and that cell infiltration is more severe than in dietary gizzard erosions.

Sixteen cesarean-derived, colostrum-deprived piglets were inoculated intranasally with porcine circovirus type 2 (PCV2), originally isolated from a pig affected with postweaning multisystemic wasting syndrome (PMWS). At 1 day postinoculation (PI), 3 of the 5 piglets in the uninoculated control group were moved to the room of inoculated piglets for contact exposure. Porcine circovirus type 2 was detected by polymerase chain reaction (PCR) in swabs from inoculated piglets from 1 day PI and from contact piglets from 2 days after cohabitation. Porcine circovirus type 2 was also detected in all serum samples but not in control piglets 7 days PI. Until the end of study, PCV2 was detected in swabs and serum samples by PCR but not in the control piglets. One inoculated piglet died suddenly without clinical signs 19 days PI. Beginning at 14 days PI, 5 piglets, including 1 contact piglet, had clinical signs of depression, anorexia, and icterus, and 1 inoculated piglet died 21 days PI. Most of the piglets exhibiting the above clinical signs became moribund and were necropsied 21 and 28 days PI. In the piglets that showed clinical signs, gross lesions, including icterus of liver and hemorrhage in stomach, and typical histopathological lesions of PMWS, such as lymphoid depletion and basophilic intracytoplasmic inclusion bodies in lymph nodes and other tissues, were observed. Porcine circovirus type 2 was detected by PCR in all tissue samples except in those of the control piglets. Porcine circovirus type 2 was recovered from several tissue samples of the piglets necropsied until 35 days PI. In particular, PCV2 was recovered in high titer from most of the tissue samples of the piglets exhibiting clinical signs. Serum antibody against PCV2 was mostly detected in inoculated piglets and in contact piglets 14 and 21 days PI by an indirect fluorescence antibody test but was not detected in the piglets exhibiting clinical signs until 28 days PI. These results indicate that PCV2 was able to induce clinical PMWS in the absence of other swine pathogens and that there were significant differences in both the quantitative PCV2 distribution in tissues and the antibody response between the piglets that were infected and developed PMWS and those that were infected but remained healthy.

The expression of type-1 Lewis antigens on erythrocytes and in digestive organs is determined by a Lewis type alpha(1,3/1, 4)-fucosyltransferase (Lewis enzyme) encoded by the Fuc-TIII gene ( FUT3 gene; Lewis gene). We have classified the Lewis alleles in the Japanese population into four types, the wild-type allele ( Le ) and three mutated alleles, i.e., le1, which has missense mutations T59G and G508A, le2, which has T59G and T1067A, and le3, which has only T59G. Here we carried out an extensive study on the biological properties of the three mutant Lewis enzymes, the le1, le2, and le3 enzymes, using native tissues and obtained the following results. (1) In in vivo and in vitro experiments, the le1 and le2 enzymes were found to be susceptible to protease digestion probably because the one missense mutation in the catalytic domains, i.e., Gly170 to Ser in the le1 enzyme and Ile356 to Lys in the le2 enzyme, makes the three-dimensional structures of the enzymesunstable, while the le3 and wild-type Lewis enzymes wereresistant to protease digestion. (2) The le1 and le2 enzymes cannot synthesize type 1 Lewis antigens on either glycolipids or mucins. The le3 enzyme cannot synthesize Lewis-active glycolipids, which result in the Lewis antigen-negative phenotype of erythrocytes, while it can synthesize Lewis antigens on mucins in normal and cancerous colon tissues. The missense mutation, Leu20 to Arg, in the transmembrane domain reduces retention of the le3 enzyme in the Golgi membrane resulting in an apparent reduction of enzyme activity as revealed by the lack of Lewis antigen synthesis. (3) The Lewis gene dosage actually has effects in vivo on the amount of the Lewis enzyme, its activity, and finally the amounts of Lewis carbohydrate antigens. This is the first article that clearly demonstrates the gene dosage effects on the amount of the glycosyltransferase protein, its activity, and the amounts of carbohydrate products in vivo.

The Economic Load Dispatching (ELD) has been and is one of the key functions to be carried out in the Central Dispatching Office. Nowadays, owing to the increase of nuclear power plants, coal- & oil-fired power plants are asked to play a more important role in the ELD satisfying various operating constraints. This paper presents a unique algorithm for on-line ELD of 2-level scheme. The method, forecasting the total load demand P over one hour, handles changing the number of coal-mills & burners and output zone switching in terms of Advance-Dispatching-Time and Min-Zone-Time. The 2-level structure of the proposed ELD method is efficient computationally and is suited for on-line use. A more friendly procedure of the output zone switching is also tried using a small set of knowledge. The proposed method is being tested in the field aiming at the advanced automated operation in a utility.

An anti-H antibody was demonstrated to be produced in egg yolk as well as in serum of hens which were immunized with human type O red blood cells. The antibody in egg yolk was isolated with polyethylene glycol and ethanol and was purified by affinity chromatography using immunoadsorbant beads immobilized with H type 2 hapten (Fucai2Ga1β14GlcNAcβ). Hemagglutination reaction of the antibody from egg yolk was inhibited by human saliva samples from secretor types irrespective of their ABO blood types, and by immunoadsorbant beads which contain Fucal2Galβ structures.

An affinity column containing L-fucose specific Aleuria aurantia lectin was used for the efficient separation of tumor-associated antigens. Five of six glycoconjugates antigens tested, carcinoembryonic antigen (CEA), CA19-9, sialyl Lewis X-i, DU-PAN-2 and CA125, bound to the affinity gel and eluted in high yield with 20 mM L-fucose, but α-fetoprotein, which is known to contain fucose in the carbohydrate chain, passed through the column. This column was also proved to be useful for group separation of CA19-9, sialyl Lewis X-i and Leb antigens from a serum sample with gastric cancer.

Antibody to the carbohydrate moiety of T antigen was developed. The synthetic antigen (Galβ1→3 GalNAcα1→OC6H4 N=N-BSA) was prepared by coupling the diazonium salt of the disaccharide derivative Galβl→3 GalNAcα→OC6H H4NH2 (o) with bovine serum albumin. Specificity of the antibody produced was examined with structurally related synthetic saccharides using the enzyme immunoassay technique. The presence of a glycosyl group at 0-6 of either the Gal or the GalNAc residue of the disaccharide Galβl→3 GalNAc did not prevent binding of the antisera to the saccharide moiety. However, the antisera did not bind either the trisaccharide moiety NeuAc2→3 Galβ→3 GalNAcαl→OC6H4NO2 (O) or GlcNAcβl→3 Galβl→3 GalNAc α OBn. These observations indicate that antibody approach to the antigen is to the 0-3 side of the terminal galactose in the disaccharide Galβl→3 GalNAc. We have also observed that the antibody prefers Galβl→3 GalNAcα1→ to Galβl→3 GalNAcβl → disaccharide derivatives in its binding capacity., The antibody was found to bind natural T antigen present on neuraminidase-treated red blood cells and, by immunohistochemical analysis, it was found to bind to naturally ocurring T antigen on breast tumor cells.

Antibody to chemically synthesized Fuc α 1→;3 Gal-1→;0C6H4N = N bovine serum albumin was developed. Specificity analysis using related synthetic saccharides was performed. The antisera were shown to be specific for both sugars in the disaccharide, to the 1→;3 positional linkage and to the α anomeric configuration. Immunohistochemical staining was performed on benign disease and adenocarcinoma tissue of the colon. 5/8 of the benign colon polyp tissue and 4/6 of the colonic adenocarcinoma tissue showed strong granular cytoplasmic staining of the epithelia cells lining the ducts. None of the six normal colon tissues tested showed any reactivity. Immunohistochemical staining was also performed on tissue from benign disease and from carcinoma of the mammary gland. 2/5 of the tissue from benign disease of the breast and 3/6 of the tissue from breast carcinoma showed strong granular cytoplasmic staining of the ductal epithelial cells.

Abstract: A new procedure for in vitro propagation of orchids belonging to the genus Phalaenopsis was developed. In contrast to commonly employed propagation methods that make use of leaf, root, or shoot tip tissues, we have used elongated stems of 6-benzyladenine-induced young seedlings as starting material for propagation. The elongated stem consisted of several nodes of which top nodes were used for cyclic propagation of new explants and the middle nodes for producing shoots or multiple adventitious buds. The whole procedure of proliferation could be completed within 7 months, and about 2,300 plantlets were produced from a single induced stem in a single year. This method may be used for propagation of seedlings in the case of lack of seeds in orchid breeding or for propagation of vegetative buds developed on flower stalks of rare orchid varieties when available flower stalks are limited. It may also have great potential for the propagation of wild threatened orchid species.

Human α(1,3/1,4)fucosyltransferase (FucT-III) genes were cloned from genomic DNAs of seven Lewis negative (Le(a- b-)) individuals. Restriction mapping analyses revealed that an additional Pvu II site was created in the FucT-III genes from Lewis negative individuals. Four of these individuals were shown to be heterozygous for the Pvu II site and three were homozygous for it. The Pvu II site was produced by a single base substitution at the 508 nucletide of coding sequence, G to A, resulting in the amino acid substitution, Gly to Ser at position 170. Another single base substitution, resulting in the change of Asp to Ala at position 336, was observed in the Lewis negative FucT-III gene which did not have the Pvu II site. The FucT-III enzyme was inactivated by these two mutations resulting in a single amino acid substitution in the catalytic region. All FucT-III genes, regardless of the Pvu II site, from Lewis negative individuals had a single base substitution, the T at position 59 to G, resulting in the change of Leu to Arg at position 20. This mutation, however, was not responsible for FucT-III gene inactivation.Copyright 1993, 1999 Academic Press

Objective To investigate somesthetic functions of the perisylvian cortex. Methods Somatosensory evoked magnetic fields (SEFs) and somatosensory evoked potentials (SEPs) of the perisylvian cortex were recorded directly from subdural electrodes in a patient with a left frontal brain tumour. Results The most prominent SEP components after electrical stimulation of the right and left hands and the right foot were double peaked negativity recorded just above the sylvian fissure (latency 80 to 150 ms), respectively (N1a and N1b). Generator sources for the magnetoencephalographic counterparts of those peaks (N1a(m) and N1b(m)) were both localised at the upper bank of the sylvian fissure, and those of N1a(m) were more anteromedially located than those of N1b(m). Conclusions These findings suggest the existence of at least two separate somatosensory areas within the human perisylvian cortex.

The expression of Lewis histo-blood group antigen is determined by the Lewis type alpha(1,3/1,4) fucosyltransferase encoded in Fuc-TIII gene (Le gene) on chromosome 19. Weak hemagglutination reactions are often observed in the Lewis blood typing to cause false-negative reactions. The level of CA19-9 tumor marker, defined as sialylated Le(a) (sLe(a)), is influenced by the Lewis blood phenotype. The sLe(a) antigen is also considered to play a role in hematogenous metastasis of tumor cells. The accurate genotyping of Fuc-TIII gene would be, therefore, necessary for solving above questions. In addition to the two missense mutations previously reported by us, i.e. the T59G and the G508A, further sequence analyses revealed a new single base substitution, the T1067A, changing the Ile356 to Lys in the catalytic region. Polymerase chain reaction-restriction fragment length polymorphism methods were developed for the detection of these three missense mutations and applied in 15 Lewis negative (le/le) and 65 Lewis positive individuals (Le/Le or Le/le). These methods were found to be useful for genetic diagnosis for the Lewis histo-blood typing. All le alleles had the T59G mutation, whereas none of the Le alleles did. The le alleles were divided in two types, le1, having the G508A mutation, and le2, having the T1067A mutation. The T1067A mutation reduced the enzyme activity less than 10%, whereas the G508A mutation in the catalytic domain made the enzyme completely inactive. The frequency of occurrence of Le, le1, and le2 in the Japanese population by random sampling study was found to be 66, 30, and 4%, respectively.

The karyotype of a lungless salamander,Onychodactylus fischeri, from Korea was analyzed and compared with that of the Japanese congeneric species,O. japonicus. In both species the diploid karyotype consists of78chromosomes, including 6 pairs of large chromosomes, 6 pairs of medium-sized ones, and the remaining 27 pairs of microchromosomes. The chromosome number ofO. fischeri, 2n=78, is, like that ofO. japonicus, the largest so far reported in the order Urodela. C-banding showed that constitutive heterochromatin inO. fischeriwas mainly in the centromeric regions and near the secondary constrictions of the large chromosomes. AgNO3-bands were located in the secondary constrictions associated with C-band heterochromatin.

Summary: (agr1rarr3)-l-Fucosyltransferase activity was measured in serum samples from 90 gastric cancer patients, 10 patients with benign diseases and 100 healthy controls. The enzyme activity was significantly elevated in the serum samples of patients with cancer compared to those from patients with benign diseases (P<0.01) and healthy controls (P<0.001). The elevation of the enzyme activity was found to correlate strongly with the clinical stage of disease. The sensitivity of (agr1rarr3)-l-fucosyltransferase was also demonstrated to be high in comparison with the tumor-associated antigens, such as carcinoembryonic antigen and sialylated Lewis X-i. Follow-up studies of (agr1rarr3)-l-fucosyltransferase in 11 cancer patients with disease at different stages showed that the enzyme activity could be useful for monitoring the postsurgical course of the disease. These results suggest that (agr1rarr3)-l-fucosyltransferase activity has a clinically important potential as a tumor marker in gastric cancer.

A new capsiate-like substance, named nordihydrocapsiate (1), has been isolated from the fruits of a nonpungent cultivar, CH-19 Sweet, of pepper (Capsicum annuum). The structure of 1 was determined to be 4-hydroxy-3-methoxybenzyl 7-methyloctanoate by spectroscopic methods.

Planarians have strong regenerative ability derived from their pluripotent stem cells, which are distributed throughout their body. If we cut a planarian into many pieces, nearly all fragments can regenerate the entire structure of the body (Yazawa et al., 2009; Agata and Umesono, 2008; Kobayashi et al., 2007; Agata et al., 2003; Ogawa et al., 2002; Cebrià et al., 2002). How do the stem cells recognize the position where they should regenerate a brain, eyes or a pharynx? How can they recognize the body polarity? Here we will summarize the growth signaling systems involved in stem cell regulation during planarian regeneration.Yazawa, S., Umesono, Y., Hayashi, T., Tarui H., Agata, K., 2009. Planarian Hedgehog/Patchedestablishes anteroposterior polarity by regulating Wnt signaling. Proc. Natl. Acad. Sci. USA 106, 22329–22334.Agata, K., Umesono, Y., 2008. Brain regeneration from pluripotent stem cells in planarian. Philos. Trans. R. Soc. Lond. B Biol. Sci. 363, 2071–2078.Kobayashi, C., Saito, Y., Ogawa K., Agata, K., 2007. Wnt signaling is required for antero-posterior patterning of the planarian brain. Dev. Biol. 306, 714–724.Agata, K., Tanaka, T., Kobayashi, C., Kato K., Saito, Y., 2003. Intercalary regeneration in planarian. Dev. Dyn. 226, 308–316.Ogawa, K., Ishihara, S., Mineta, K., Nakazawa, M., Ikeo, K., Gojobori, T., Watanabe K., Agata, K., 2002. Induction of a noggin-like gene by ectopic D–V interaction during planarian regeneration. Dev. Biol. 250, 59–70.Cebrià, F., Kobayashi, C., Nakazawa, M., Mineta, K., Ikeo, K., Gojobori, T., Ito, M., Taira, M., Sánchez Alvarado, A., Agata, K., 2002. FGFR-related gene nou-darakerestricts brain tissues to the head region of planarians. Nature 419, 620–624.