The Association of Ascorbate and Ascorbate Oxidase in the Apoplast with IAA-Enhanced Elongation of Epicotyls from Vigna angularis

The level of (ascorbic acid (AA) plus dehydroascorbic acid (DHA))and the ratio of the level of AA to that of AA plus DHA in intercellularwashing fluid (IWF) of epicotyl segments from Vigna angularisdecreased from 2.8±0.3 to 1.2±0.5nmol (g fr wt)^–1and from 0.23±0.03 to 0.13±0.01, respectively,after incubation of the segments without IAA for 20 h at 27°C.However, these values changed to 5.3±1.7 nmol (g fr wt)^–1and 0.07±0.05 after incubation with 0.1 mM IAA. The activityof cell wall-bound ascorbate oxidase increased by about 20%and 70% after incubation without IAA and with IAA, respectively.However, the activity of cell wall-bound peroxidase was notaffected by incubation with or without IAA. The activities ofascorbate oxidase and peroxidase in IWF decreased by about 85and 75% after incubation without IAA. IAA did not affect thesedecreases to any great extent. A lignin-like compound was formedduring the incubation of epicotyl segments in the absence ofIAA. Formation of this compound was inhibited by IAA. The resultssuggest that one of the causes of the enhancement of elongationgrowth by IAA is the inhibition of peroxidase-dependent lignificationas a result of increases in levels of AA and DHA and in ascorbateoxidase activity. (Received August 16, 1993; Accepted December 6, 1993)     

Adaptations for feeding on rock surfaces and sandy sediment by the fiddler crabs (Brachyura: Ocypodidae) Uca tetragonon(Herbst, 1790) and Uca vocans(Linnaeus, 1758)

Two species of fiddler crab, Uca tetragonon(Herbst, 1790) and Uca vocans(Linnaeus, 1758), which belong to the subgenus Gelasimus, dwell on rocky shores and muddy–sandy tidal flats, respectively, in Phuket Is., Thailand. We investigated their feeding ecology in relation to the morphology of their feeding organs: minor food-handling chelipeds and maxillipeds. U. tetragononfed chiefly on rocks covered by filamentous green algae. U. vocansfed on the emerged sand and in shallow water along the shoreline and in pools. While feeding, both crabs made sand pellets beneath their mouthparts and discarded them, indicating that they divided the matter scooped up with their minor chelipeds into edible and inedible fractions by using the maxillipeds in the water passing through their buccal cavity. The morphology of maxillipeds hardly differed between the two species, which means that both species are flotation-feeders. The morphology of their minor chelipeds, however, differed: the tips of the dactyl and pollex were flat in U. tetragononand pointed in U. vocans.When the minor cheliped was closed, U. tetragononhad a hemispherical space in the distal one-fourth of the gape, which was closed by the framing keratin layers and a few setae of the dactyl and pollex. On the other hand, U. vocanshad an ellipsoidal space in the distal half of the gape. We consider these morphological characters to be adaptations to the different feeding substrates for retaining more food-laden sediment. We discuss the role of the setae on the minor chelipeds on the basis of the morphological differences between populations of U. tetragononin Phuket Is. and East Africa where the crab inhabits muddy–sandy tidal flats.     

Inhibiting Effect of Auxin on Glucosamine Incorporation into Cell Walls of Rice Coleoptiles

Auxin induced growth and decreased the hexosamine content ofthe cell walls of rice coleoptile sections. Indole-3-aceticacid (IAA) at 10^–5 M inhibited the incorporation of ¹⁴C-glucosamineinto the cell walls. IAA did not affect the ¹⁴C-incorporationinto the cytoplasm, while inhibitors of glycoprotein synthesis,unicamycin and monensin, suppressed the incorporation into boththe cytoplasm and the cell walls. The radioactivity due to labeledglucosamine in the cell walls increased during the chase, butthis increase was inhibited by IAA. Among the cell wall fractions,the increase in radioactivity and its inhibition by IAA wereconspicuous in the hemicellulose I fraction. The inhibitoryeffect of IAA on glucosamine incorporation into the cell wallswas observed even in the presence of 0.15 M mannitol solutionwhich completely suppressed the IAA-induced growth. These resultssuggest that auxin induces growth at least partly by inhibitingthe transport of asparagine-linked glycoproteins from the cytoplasmto the cell walls. ¹ Present address: Department of Biology, Faculty of Science,Osaka City University, Sumiyoshi-ku, Osaka 558, Japan (Received July 23, 1986; Accepted December 22, 1986)     

Chloroplast genome evolution in the genus Avena

The genus Avena contains five different chloroplast genomes, I-V. A physical map of chloroplast (ct) DNA of Avena sativa (type I chloroplast genome) was constructed using three restriction endonucleases, PstI, SalI and SmaI. This genome is ca. 135.5 kbp in size, and contains two inverted repeats of ca. 22.5 kbp each, separated by a large (ca. 79.0 kbp) and small (ca. 12.5 kbp) single copy region. The rbcL gene which codes for the large subunit of ribulose 1,5-bisphosphate carboxylase, was located in the map. Restriction fragment patterns of all five chloroplast genomes were compared, and among them five fragment size and five restriction site mutations were disclosed. Four site mutations were found in two or more chloroplast genomes, the other site and five fragment size mutations were specific to one or another of the chloroplast genomes. A dendrogram showing phylogenetic relationships among the five chloroplast genomes, based on the distribution of the common and specific mutations among them, indicates that chloroplast genome divergence characterized by three restriction site mutations occurred first between two diploid groups, each carrying A and C genome (nuclear), respectively, followed by further speciation in each group.     

Identification of a novel nifH-like (frxC) protein in chloroplasts of the liverwort Marchantia polymorpha

The frxC gene, one of the unidentified open reading frames present in liverwort chloroplast DNA, shows significant homology with the nifH genes coding for the Fe protein, a component of the nitrogenase complex (Ohyama et al., 1986, Nature 322: 572–574). A truncated form of the frxC gene was designed to be over-expressed in Escherichia coli and an antibody against this protein was prepared using the purified product as an antigen. This antibody reacted with a protein in the soluble fraction of liverwort chloroplasts, which had an apparent molecular weight of 31 000, as revealed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, in good agreement with a putative molecular weight of 31945 deduced from the DNA sequence of the frxC gene. In a competitive inhibition experiment, the antigenicity of this protein was indicated to be similar to that of the over-expressed protein in E. coli. Therefore, we concluded that the frxC gene was expressed in liverwort chloroplasts and that its product existed in a soluble form. The molecular weight of the frxC protein was approximately 67 000, as estimated by gel filtration chromatography, indicating that the frxC protein may exist as a dimer of two identical polypeptides analogous to the Fe protein of nitrogenase. The results obtained from affinity chromatography supported the possibility that the frxC protein, which possesses a ATP-binding sequence in its N-terminal region that is conserved among various other ATP-binding proteins, has the ability to bind ATP.     

Determination of the primary structure of Paim II, an alpha-amylase inhibitor from Streptomyces coruchorushii, by high-performance tandem mass spectrometry

This study indicates one of the advantages of tandem mass spectrometry; the primary structures of proteins with little structural difference can be determined by using tandem mass spectrometry without prior purification of each component. The primary structure of Paim II, a protein alpha-amylase inhibitor from Streptomyces coruchorushii, was determined by using tandem mass spectrometry. Paim II consists of two component proteins with ragged N-terminus, and was sequenced on the basis of the structure of Paim I, an analogous alpha-amylase inhibitor from the same natural origin.     

Comparison of glycoconjugates at the surface of developing type II pneumocytes and Clara cells

Summary The apical surface coat of type II pneumocytes and Clara cells in pre- and post-natal rat lung was examined with lectin histochemical methods. Lectins fromHelix pomatia (HPA), peanut (PNA) andMaclura pomifera (MPA) were conjugated with horseradish peroxidase and used to stain paraffin sections of fixed lung with or without certain pre-treatments. HPA and MPA were observed to react with almost all type II pneumocytes at postnatal day 1. Type II pneumocytes that stained with a sialidase—PNA sequence increased from a few positive cells at postnatal day 5 to many in the adult. It has been reported that the surface coat of type II pneumocytes closely resembles that of Clara cells in its staining with histochemical methods employing cationic dyes or lectins including MPA and PNA. However, staining with HPA, especially after periodic acid oxidation, revealed many type II pneumocytes with strong reactivity but showed only a few Clara cells that were faintly positive. HPA also stained alveolar macrophages. The HPA affinity of macrophages, however, was labile to oxidation with periodic acid or galactose oxidase unlike that of type II pneumocytes. This difference suggests that HPA recognizes more than one type of sugar structure.To whom all correspondence and reprint requests should be addressed.     

Paradoxical enhancement of interleukin-2-mediated cytotoxicity against K562 cells by addition of a low dose of methotrexate

Summary In vitro effects of methotrexate (MTX) on interleukin-2(IL-2)-mediated cytotoxicity of peripheral blood mononuclear cells (PBMC) were studied. PBMC were incubated with human recombinant IL-2 (25 U/ml) for 72 h; during the last 24 h, various concentrations (10 pM–1 µM) of MTX were added to the culture. Cytotoxicity against k562 cells was measured by a 4-h⁵¹Cr-release assay. The IL-2-mediated cytotoxicity was paradoxically increased at around a concentration (10 nM) MTX. Such a low concentration of MTX showed no anti-proliferative effect on cell growth. This enhancement with 10 nM MTX was shown only in an E-rosette⁺ (E⁺) population, but not in E-rosette^– (E^–). In addition, when E⁺ cells were treated with an anti-CD16 monoclonal antibody plus complement after incubation with IL-2 and MTX, MTX-induced enhancement was lost, suggesting that an E⁺CD16⁺ cell population was mainly involved in this augmentation. Positively sorted E⁺CD16⁺ cells showed similar enhancement of cytotoxicity after treatment with IL-2 plus MTX. On the other hand, MTX treatment did not show the phenotypical changes including of the E⁺CD16⁺ cells, indicating that this treatment did not affect the differentiation and proliferation of the specific cell subset. Our results indicate that a low dose of MTX could have a role in the regulation of immunological anti-cancer surveillance systems through the natural killer and lymphokine-activated cytotoxic cells.This work was supported in part by a Grant-in-Aid for Cancer Research (1–10) from the Ministry of Health and Welfare in Japan     

The augmentation of tumor-specific immunity using haptenic muramyl dipeptide (MDP) derivatives

Summary A previous paper has demonstrated that enhanced tumor-specific immunity could be induced by priming mice with Bacillus Calmette Guerin (BCG) and subsequently immunizing them with syngeneic tumor cells modified with BCG-cross-reactive muramyl dipeptide (MDP) hapten [15]. The present study establishes a tumorspecific immunotherapy protocol for a murine chronic leukemia based on the above T-T cell collaboration between antitumor effector T cells and anti-MDP hapten helper T cells induced by BCG priming. BALB/c mice which had been primed to BCG were injected intravenously (i.v.) with viable, syngeneic BCL1 leukemia cells. One week later, these mice were immunized intraperitoneally (i.p.) with unmodified or MDP hapten-modified, 10,000 R X-irradiated BCL1 cells, followed by 4 booster immunizations at 5-day intervals. The administration of unmodified BCL1 tumor cells into BCG-primed mice failed to prevent them from tumor death due to the persistent growth of preinjected BCL1 cells. In contrast, the immunization of BCG-primed, BCL1 leukemia-cell-bearing mice with MDP-modified BCL1 cells resulted in a high growth inhibition of leukemia cells and protection of these mice from death by leukemia. It was also revealed that potent tumorspecific, T-cell-mediated immunity was generated in mice which survived in this immunotherapy model. Thus, these results indicate that administration of MDP hapten-modified, syngeneic leukemia cells into leukemia-bearing mice which have been primed with BCG results in potent tumor-specific, T-cell-mediated immunity attributable to preventing the growth of disseminated leukemic cells.This work was supported by a Grant-in-Aid for the Special Project Cancer-Bioscience from the Ministry of Education, Science, and Culture, Japan Abbreviations used: TATA, tumor-associated transplantation antigens; MDP, muramyl dipeptide; MTP, muramyl tripeptide; BCG, Bacillus Calmette Guerin