A 5-methyltryptophan resistant rice mutant,MTR1, selected in tissue culture

Summary Cell lines resistant to tryptophan analogue 5-methyltryptophan (5MT) were selected in seed-derived calli of Oryza sativa L. var. Norin 8. Plants were regenerated (R₁ from one selected callus line (MTR1). In three out of the six R₁ plants, 5MT resistance was inherited in the R₂ and R₃ generations as a dominant nuclear mutation. Segregation ratios in the progeny of heterozygous plants were 11. Morphological and fertility variation seen in some of the R₂ plants were not correlated with 5-methyltryptophan resistance. Resistance in the MTR1 callus was due to the accumulation of high levels of free tryptophan (87-fold) that was associated with an increase in free phenylalanine content (9-fold). The leaves of resistant plants also contained elevated levels of free tryptophan and phenylalanine.Abbreviations 2,4-D 2,4-dichlorophenoxyacetic acid - MS Murashige and Skoog (1962) basal medium - 5MT D,L-5-methyltryptophan - phe phenylalanine - trp tryptophan - tyr tyrosine     

Breeding synchrony and spawning intervals in the temperate damselfishChromis notata

Synopsis The reproductive behavior of damselfish,Chromis notata, was studied at Mukaishima Island, Seto Inland Sea of Japan. Spawning occur synchronously among the fish in the study area except during a part of the breeding season. Neither spawnings nor hatchings, however, coincide with any particular phase of the lunar or tidal cycle. The intervals between the spawning peaks vary considerably during the breeding season. These results support the hypothesis that synchrony of breeding is facilitated by social interactions among the fish of a local area. The male inter-spawning intervals, and even the intervals between last spawning and next nesting, are longer than the female inter-spawning intervals in the beginning of breeding season. This delay of male spawning is attributed to the long incubation period and may have an influence on the male reproductive success.     

Polysaccharides of the cellular slime mold II. Change of intra- and extracellular polysaccharides during growth phase of Dictyostelium discoideum NC-4

The three intra- and extracellular polysaccharide fractions were isolated during the growth phase of Dictyostelium discoideum NC-4, and the change in content of component sugars of four fractions during the culture period was examined. Myxamoebae most extensively contain a polysaccharide fraction extracted with phenol-water (polysaccharide fraction I) in a quantity of about 15–23% per dry cell. After 15 h the uronic acid formed in the polysaccharide fraction I, and the cell, could be aggregated. The glucosamine content in the polysaccharide fraction I reached a maximum as the myxamoebae entered the exponential phase, and a large amount of galactose was produced as the cell entered the stationary phase. The phenol-water extract from the cells of the stationary phase was reacted with concanavalin-A.     

Induction of gene expression for nitric oxide synthase by immunomodulating drugs in the RAW264.7 murine macrophage cell line

 We have elucidated the direct effects of PSK (a protein-bound polysaccharide) and OK-432 (a streptococcal preparation), both immunomodulating drugs, on the gene expression for an inducible nitric oxide synthase and on the production of nitric oxide (NO) in the RAW264.7 murine macrophage cell line. As determined by northern blot analysis, both immunomodulating drugs were potent inducers of gene expression for inducible NO synthase when cells were costimulated with interferon-γ (IFNγ). Expression of mRNA for the enzyme occurred in a dose-dependent manner after 3 h, when 10 – 50 μg/ml PSK or 0.001 – 1 KE/ml OK-432 was used. Furthermore, NO was also produced in response to these drugs, as detected by the Griess reagent reaction. The enhancement of NO synthesis was thought to be mediated, in part, through tumor necrosis factor α (TNFα) induction by these agents, since a neutralizing antibody to TNFα significantly suppressed NO production in RAW264.7 cells stimulated with PSK or OK432 in combination with IFNγ. We speculate that NO production may play a role in tumoricidal and microbicidal activities of PSK or OK-432 in vivo. Received: 9 August 1995 / Accepted: 1 April 1996     

Molecularly imprinted polymer-assisted refolding of lysozyme

For production of active proteins using heterologous expression systems, refolding of proteins from inclusion bodies often creates a bottleneck due to its poor yield. In this study, we show that molecularly imprinted polymer (MIP) toward native lysozyme promotes the folding of chemically denatured lysozyme. The MIP, which was prepared with 1 M acrylamide, 1 M methacrylic acid, 1 M 2-(dimethylamino)ethyl methacrylate, and 5 mg/mL lysozyme, successfully promoted the refolding of lysozyme, whereas the non-imprinted polymer did not. The refolding yield of 90% was achieved when 15 mg of the MIP was added to 0.3 mg of the unfolded lysozyme. The parallel relationship between the refolding yield and the binding capacity of the MIP suggests that MIP promotes refolding through shifting the folding equilibrium toward the native form by binding the refolded protein.     

Eight new species of the genus <Emphasis Type="Italic">Mycena</Emphasis> from central Honshu,Japan

Eight new species of the genus Mycena (Agaricales) found on dead leaves or twigs in warm temperate lowland forests are described and illustrated from central Honshu, Japan: (1) Mycena fonticola sp. nov. (section Fragilipedes); (2) Mycena intersecta sp. nov. (section Fragilipedes); (3) Mycena lanuginosa sp. nov. (section Fragilipedes); (4) Mycena mustea sp. nov. (section Fragilipedes); (5) Mycena multiplicata sp. nov. (section Mycena); (6) Mycena nidificata sp. nov. (section Hiemales); (7) Mycena fuscoaurantiaca sp. nov. (section Fragilipedes); and (8) Mycena clariviolacea sp. nov. (section Fragilipedes).     

Five new species of the Boletaceae from Japan

Five new species of the Boletaceae (Agaricales) from Japan are described and illustrated: (1) Boletus bannaensis sp. nov. (section Luridi), forming a grayish-brown pileus and rufescent flesh, found in subtropical evergreen broad-leaved forests; (2) Leccinum rhodoporosum sp. nov., forming discolorous red pores, a whitish stipe covered overall with violet-brown to blackish-brown furfuraceous scales and fusoid-cylindrical brown basidiospores, found in subtropical evergreen broad-leaved forests or warm-temperate Quercus-Pinus forests; (3) Pulveroboletus brunneoscabrosus sp. nov., forming a lemon-yellow pulverulent basidiomata covered overall with orange to brownish-orange appressed scales, found in subtropical to warm temperate evergreen broad-leaved forests; (4) Rubinoboletus monstrosus sp. nov., forming a brownish-orange to yellowish-brown pileus and a very short, nonreticulate, hollow stipe, found in subtropical evergreen broad-leaved forests; and (5) Tylopilus fuligineoviolaceus sp. nov., having a deep violet to blackish-brown pileus and brunnescent hymenophore, found in warm temperate Quercus-Castanopsis forests.     

Higher order structure contributes to specific differences in redox potential and electron transfer efficiency of root and leaf ferredoxins

Plant type ferredoxin (Fd) is a small [2Fe-2S] cluster containing electron-transfer protein with a highly negative redox potential. Higher plants contain different iso-protein types of Fd in roots and leaves, reflecting the difference in redox cascades between these two tissues. We have combined subdomains of leaf and root Fds in recombinant chimeras, to examine structural effects and the relationship between groups of residues on redox potential, electron transfer, and protein-protein interactions. All chimeras had redox potentials that were intermediate to the wild type leaf and root Fds. Surprisingly, the largest differences resulted from exchange of the N-terminus, the region farthest from the redox center. Homology modeling and energy minimization calculations suggest that the N-terminal chimeras may indirectly influence redox potentials by structurally perturbing the active site. Measurements of electron transport and protein interaction indicate that synergistic interaction between the C- and N-terminal of root Fd bestows a specific high affinity for accepting electrons in the root type electron cascade, and that there is discrimination against photosynthetic electron donation to root Fd based on the C-terminus of the molecule. Taken together, the experimental and computational studies support a model in which higher order structure contributes to iso-protein specific interaction and electron-transfer properties.     

Utilization of Methyl Proton Resonances in Cross-Saturation Measurement for Determining the Interfaces of Large Protein–Protein Complexes

Cross-saturation experiments allow the identification of the contact residues of large protein complexes (MW>50 K) more rigorously than conventional NMR approaches which involve chemical shift perturbations and hydrogen-deuterium exchange experiments [Takahashi et al. (2000) Nat. Struct. Biol., 7, 220–223]. In the amide proton-based cross-saturation experiment, the combined use of high deuteration levels for non-exchangeable protons of the ligand protein and a solvent with a low concentration of ¹H₂O greatly enhanced the selectivity of the intermolecular cross-saturation phenomenon. Unfortunately, experimental limitations caused losses in sensitivity. Furthermore, since main chain amide protons are not generally exposed to solvent, the efficiency of the saturation transfer directed to the main chain amide protons is not very high. Here we propose an alternative cross-saturation experiment which utilizes the methyl protons of the side chains of the ligand protein. Owing to the fast internal rotation along the methyl axis, we theoretically and experimentally demonstrated the enhanced efficiency of this approach. The methyl-utilizing cross-saturation experiment has clear advantages in sensitivity and saturation transfer efficiency over the amide proton-based approach. Electronic supplementary material Electronic supplementary material is available for this article at and accessible for authorised users.