Molecular characterization of MHC class I and beta-2 microglobulin in a clonal strain of ginbuna crucian carp, Carassius auratus langsdorfii

Clonal ginbuna crucian carp is, a naturally gynogenetic fish, and is a useful model animal for studying T-cell-mediated immunity. To gain molecular information on MHC class I molecules from this species, we have identified four types of MHC class I (caauUA-S3n, caauUF-S3n, caauZE-S3n, and caauZB-S3n) and five beta 2-microglobulin (β(2)m) (caauβ2m-1a, caauβ2m-1b, caauβ2m-2, caauβ2m-3a and caauβ2m-3b) by an expressed sequence tag (EST) analysis and using homology cloning with degenerated primers. Like UA class I genes in other cyprinid fish, the caauUA-S3n shows features of classical MHC class I, such as conservation of all key amino acids interacting with antigenic peptides, and ubiquitous tissue expression. A phylogenetic analysis shows that the β(2)m-1 and β(2)m-2 isoforms are clustered with those of other cyprinid fishes, while β(2)m-3 isoforms make a cluster that is separated from a common ancestor of salmonid and cyprinid fishes. This finding suggests that the β(2)m isoforms of ginbuna cruician carp comprise two lineages and may possess different functions. The MHC class I and β(2)m sequences from one clonal strain will facilitate our understanding of the interaction of MHC class I with β(2)m in teleosts.     

Ski slope vegetation at Snoqualmie Pass, Washington State, USA, and a comparison with ski slope vegetation in temperate coniferous forest zones

Ski slope vegetation at Snoqualmie Pass in Washington State, USA, was surveyed in order to identify community types and to compare it with vegetation development patterns in Japan. Ski slopes in Japan, most of which were constructed after 1960, underwent heavy land recontouring, while those at Snoqualmie Pass were constructed before 1950 with less modification. Three points apply to Japanese ski slope vegetation and differentiate these slopes from those at Snoqualmie Pass: (i) grasslands of introduced species are widespread and persistent; (ii) unvegetated patches are uncommon; and (iii) wetland vegetation has developed. These differences are mainly derived from the intensity of human impact, history of the slope and its scale: namely, ski slopes in Washington are older and larger than those in Japan. Ski slope vegetation in Washington was primarily differentiated by a soil moisture gradient. The large size of Washington ski slopes permitted the inclusion and development of wetland habitats, whereas most ski slopes in Japan are constructed on ridges and do not contain wetlands. Most introduced species in Japan are eliminated soon after seeding. In contrast, the long-term management of ski slopes decreased soil erosion and/or unvegetated patches in Washington and created relatively permanent grasslands composed of introduced species. Tsuga heterophylla and Abies amabilis were found established on the ski slopes in Washington, whereas in Japan the pioneer tree species are shade-intolerant broadleaved species. These differences may be a result of the different disturbance histories of ski slopes in the two countries. In addition, along with the conifers, early successional forbs such as Anaphalis margaritacea and Epilobium angustifolium are well established on Washington ski slopes. Results show that disturbances created by ski slope development greatly affect the vegetation, even on older, less heavily impacted ski slopes.     

Mast cells promote atherosclerosis by releasing proinflammatory cytokines

Mast cells contribute importantly to allergic and innate immune responses by releasing various preformed and newly synthesized mediators. Previous studies have shown mast cell accumulation in human atherosclerotic lesions. This report establishes the direct participation of mast cells in atherogenesis in low-density lipoprotein receptor-deficient (Ldlr(-/-)) mice. Atheromata from compound mutant Ldlr(-/-) Kit(W-sh)(/W-sh) mice showed decreased lesion size, lipid deposition, T-cell and macrophage numbers, cell proliferation and apoptosis, but increased collagen content and fibrous cap development. In vivo, adoptive transfer of syngeneic wild-type or tumor necrosis factor (TNF)-alpha-deficient mast cells restored atherogenesis to Ldlr(-/-)Kit(W-sh/W-sh) mice. Notably, neither interleukin (IL)-6- nor interferon (IFN)-gamma-deficient mast cells did so, indicating that the inhibition of atherogenesis in Ldlr(-/-)Kit(W-sh/W-sh) mice resulted from the absence of mast cells and mast cell-derived IL-6 and IFN-gamma. Compared with wild-type or TNF-alpha-deficient mast cells, those lacking IL-6 or IFN-gamma did not induce expression of proatherogenic cysteine proteinase cathepsins from vascular cells in vitro or affect cathepsin and matrix metalloproteinase activities in atherosclerotic lesions, implying that mast cell-derived IL-6 and IFN-gamma promote atherogenesis by augmenting the expression of matrix-degrading proteases. These observations establish direct participation of mast cells and mast cell-derived IL-6 and IFN-gamma in mouse atherogenesis and provide new mechanistic insight into the pathogenesis of this common disease.     

Lycoparins A-C, new alkaloids from Lycopodium casuarinoides inhibiting acetylcholinesterase

Three new Lycopodium alkaloids, lycoparins A-C (1-3), have been isolated from the club moss Lycopodium casuarinoides. Structures and stereochemistry of 1-3 were elucidated on the basis of 2D NMR correlations. Lycoparins C (3) exhibited an inhibitory activity against acetylcholinesterase, while lycoparins A (1) and B (2) did not show activity.     

Pattern of glucose-14C catabolism in barley seedlings during vernalization

The pattern of glucose catabolism in barley shoots during vernalizationwas examined using glucose-1-¹⁴C and glucose-6-¹⁴C. The pentosephosphate pathway became progressively predominant over theEmbden-Meyerhof-Parnas (EMP) pathway after germination in unvernalizedseedlings, while the EMP pathway maintained a superior positionduring the corresponding developmental stages in seedlings undervernalization. (Received August 20, 1973; )     

Callus formation and plant regeneration in various <Emphasis Type="Italic">Lilium</Emphasis> species and cultivars

Summary In researching the application of genetic transformation to lily breeding, callus formation from cultured explants and plant regeneration from induced calluses were examined in 33 Lilium genotypes, 21 species, three Asiatic hybrids, two LA hybrids, two Longiflorum hybrids, three Oriental hybrids, and two Trumpet hybrids. Seed, bulb scale, leaf, or filament explants were placed on a medium containing 4.1 μM 4-amino-3,5,6-trichloropicolinic acid (picloram; PIC) and cultured in the dark. After 2 mo., callus formation was observed in 30 genotypes, and a formation frequency of more than 50% was obtained in 24 genotypes. Bulb scale and filament explants showed great ability to form calluses, whereas seeds had poor ability. Most of the induced calluses were yellow and had a nodular appearance. When subcultured onto the same fresh medium, twofold or more increases in callus mass were obtained in 1 mo. for 15 genotypes. Callus lines showing sustained growth 1 yr after the initiation of subculture were examined for their ability to produce shoots on a medium without plant growth regulators (PGRs) and a medium containing 22 μM 6-benzyladenine (BA). Shoot regeneration was observed in all genotypes examined, and a regeneration frequency of over 80% was obtained in 20 genotypes. Initial explants used for callus induction and callus type (nodular or friable) had no effect on shoot regeneration. Most of the regenerated shoots developed into complete plantlets following their transfer to a PGR-free medium.     

Regulation of aldoxime dehydratase activity by redox-dependent change in the coordination structure of the aldoxime-heme complex

Phenylacetaldoxime dehydratase from Bacillus sp. strain OxB-1 (OxdB) catalyzes the dehydration of Z-phenylacetaldoxime (PAOx) to produce phenylacetonitrile. OxdB contains a protoheme that works as the active center of the dehydration reaction. The enzymatic activity of ferrous OxdB was 1150-fold higher than that of ferric OxdB, indicating that the ferrous heme was the active state in OxdB catalysis. Although ferric OxdB was inactive, the substrate was bound to the ferric heme iron. Electron paramagnetic resonance spectroscopy revealed that the oxygen atom of PAOx was bound to the ferric heme, whereas PAOx was bound to the ferrous heme in OxdB via the nitrogen atom of PAOx. These results show a novel mechanism by which the activity of a heme enzyme is regulated; that is, the oxidation state of the heme controls the coordination structure of a substrate-heme complex, which regulates enzymatic activity. Rapid scanning spectroscopy using stopped-flow apparatus revealed that a reaction intermediate (the PAOx-ferrous OxdB complex) showed Soret, alpha, and beta bands at 415, 555, and 524 nM, respectively. The formation of this intermediate complex was very fast, finishing within the dead time of the stopped-flow mixer (approximately 3 ms). Site-directed mutagenesis revealed that His-306 was the catalytic residue responsible for assisting the elimination of the hydrogen atom of PAOx. The pH dependence of OxdB activity suggested that another amino acid residue that assists the elimination of the OH group of PAOx would work as a catalytic residue along with His-306.     

Development of a novel immobilization method for enzymes from hyperthermophiles

Peptide tags containing tyrosines (Y-tag) were introduced at the C-terminus of a hyperthermophilic enzyme, alkaline phosphatase from Pyrococcus furiosus (PfuAP). Immobilization of the recombinant PfuAPs onto water-in-oil-in-water (W/O/W) type microcapsules was performed by an in situ polymerization method. All the recombinant PfuAPs prepared in this study were quantitatively immobilized onto microcapsules. The PfuAP-immobilized microcapsules showed no significant loss of enzymatic activity until the 5th round of assays. This result implies that the recombinant PfuAPs were covalently immobilized onto microcapsules. Immobilized PfuAP tagged with a Y-tag having the sequence GGYYY exhibited approximately a twofold higher catalytic activity compared with the wild-type PfuAP. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.