Identification of Chitinase and Osmotin-Like Protein as Actin-Binding Proteins in Suspension-Cultured Potato Cells

Cytoplasmic aggregation is an early resistance-associated eventthat is observed in potato tissues either after penetrationof an incompatible race of Phytophthora infestans, the potatolate blight fungus, or after treatment with hyphal wall components(HWC) prepared from P. infestans. In potato cells in suspensionculture, the number of cells with cytoplasmic aggregation increasedupon treatment with HWC, but such an increase was suppressedby treatment with cytochalasin D prior to treatment with HWC.This result suggested that cytoplasmic aggregation in culturedpotato cells might be connected with the association of actinfilaments. To identify the molecular basis of cytoplasmic aggregation,we purified actin and actin-related proteins by affinity chromatographyon a column of immobilized DNase I from cultured potato cellsand isolated proteins of 43 kDa, 32 kDa and 22 kDa. Analysisof the amino-terminal amino acid sequences indicated that the43 kDa, 32 kDa and 22 kDa proteins were potato actin, basicchitinase and osmotin-like protein, respectively. This conclusionwas supported by the results of Western blotting analysis ofthe 43 kDa and 32 kDa proteins with antibodies against actinand basic chitinase. Binding analysis with actin coupled toactin-specific antibodies and biotinylated actin suggested thatthe 32 kDa and 22 kDa proteins had actin-binding activity. Inaddition, examination of biomolecular interactions using anoptical biosensor confirmed the binding of chitinase to actin.These results imply the possibility that basic chitinase andosmotin-like protein might be involved in cytoplasmic aggregation,hereby participating in the potato cell's defense against attackby pathogen. (Received June 11, 1996; Accepted January 27, 1997)     

r{beta}-Glucosidase in the Indigo Plant: Intracellular Localization and Tissue Specific Expression in Leaves

rß-Glucosidase of indigo plant (Polygonum tinctorium)has a high substrate specificity for indican (indoxyl rß-D-gIu-coside).To examine the localization of this rß-glucosidase,we fractionated the cells of the leaves and analysed them im-munocytochemically.Immunoelectron micrographs with specific antibodies againstthe rßglucosidase clearly showed that the rß-glucosidasewas localized in the stroma of the chloroplasts in mesophyllcells, but not in the thylakoid membrane. Chloroplasts wereisolated from the crude ho-mogenate of the fresh leaves by Percolldensity gradient centrifugation and then subjected to suborganellarfrac-tionation. rßGlucosidase activity was specificallydetected in the stromal fraction, but not in the thylakoid membrane.This was also supported by the result of an immunoblot of thefractions with anti-rßglucosidase antibodies. Therß-gIu-cosidase was immunocytochemically localizedin the chloroplasts of mesophyll cells, but not in any chloroplastsin marginal cells of the vascular bundle or epidermal cells;ribulose 1,5-bisphosphate carboxylase (Rubisco), a typical stromalprotein, was observed in all chloroplasts in these cells. Theseresults suggest that rß-glucosidase is tissue specificin its expression in the leaves of the indigo plant. (Received April 14, 1997; Accepted July 10, 1997)     

The effect of processing of Chlorellavulgaris: K-5 on in vitro and in vivo digestibility in rats

Two experiments were done to clarify whether or not cell rupture is necessary to improve the digestibility of major components of Chlorella vulgaris: K-5. Chlorella was treated with or without high pressure homogenization (1 × 10⁸ N/m² at less than −20°C) after a heating process (100-120°C). Chlorella (air-dry matter) contained 934 g dry matter and 244 g essential amino acids (total)/kg. Chemical composition was hardly altered irrespective of the treatment. In the first experiment, pepsin digestibility of chlorella protein was determined in vitro. The cell rupture by high pressure homogenization caused a small but significant improvement in pepsin digestibility of chlorella protein compared with the control. In the second experiment, total tract apparent digestibilities of chlorella were determined in the rat. Digestibility of chlorella protein was significantly enhanced by high pressure homogenization, but the difference (88.6% vs. 87.4%, P < 0.01) due to treatment was small and similar to that observed in the in vitro experiment. These results suggested that Chlorella strain vulgaris: K-5 may be an efficient protein source even without cell rupture.     

Ribosomes from spiramycin resistant mutants of Escherichia coli Q13

Summary Mutants from Escherichia coli Q13 were selected for resistance to leucomycin, tylosin or spiramycin. Most of the mutants so selected exhibited cross resistance to all the macrolide antibiotics tested including erythromycin. A few mutants however seem to be less resistant to erythromycin. One mutant, QSP008, was highly resistant to tylosin, leucomycin and spiramycin but relatively sensitive to erythromycin. Another mutant, QSP006, was highly resistant to spiramycin but less resistant to erythromycin, tylosin and leucomycin. This selective resistance of cells to specific antibiotics could be due to the extent of conformational alteration of their ribosomes, which may be demonstrated by the extent of ¹⁴C-erythromycin binding to these ribosomes. The ribosomes from QSP008 cells were found to contain an altered 50-8 protein of the 50s ribosomal subunit, while in the ribosomes from QSP006 no such protein change could be detected by the methods used.A preliminary data of part of this work has been published (Tanaka, Teraoka, Tamaki, Watanabe, Osawa, Otaka, and Takata, 1971).     

Phenotypes represented by a mutational change in a 50s ribosomal protein component, 50-8, in Escherichia coli

Summary An erythromycin resistant (ery ^r) mutant of Escherichia coli Q13, QE107, was characterized by (1) the cross resistance of the cells to several macrolide antibiotics such as erythromycin, tylosin, spiramycin, oleandomycin and leucomycin, (2) the reduced affinity of its ribosomes to erythromycin and probably to the other macrolides mentioned above, (3) a low peptidyl transferase activity of its ribosomes and (4) an altered 50-8 protein of the 50s ribosomal subunits. These characters were always transferred together with the ery marker in the transduction experiments.Preliminary data of part of this work has been published (Tanaka, Teraoka, Tamaki, Watanabe, Osawa, Otaka and Takata, 1971).     

Control of cell division in a green alga, Ankistrodesmus gracilis

The proliferation pattern of Ankistrodesmus gracilis, a speciesof Chlorococcales, is described. Under standard conditions,various proliferation patterns; di-, tri-, tetra-, or multichotomicalcell division were observed. Light-dark diurnal rhythms, LD12 : 12 and 14 : 10 induced growth patterns which formed two-to several-ten-celled colonies, whereas the rhythms LD 16 :8 to 20 : 4 induced only the formation of two-celled colonies.These inductions were observed at a cell density of 4.0?10⁶cells/ml. Dichotomical cell division occured at a cell densityof more than 1.5?10⁷ cells/ml. No influence of self-shadingon the pattern of colony formation was detected. (Received May 30, 1974; )     

N-ethylmaleimide-resistant acyl-coenzyme A oxidase from Arthrobacter ureafaciens NBRC 12140: molecular cloning, gene expression and characterization of the recombinant enzyme

N-ethylmaleimide (NEM)-resistant acyl-coenzyme A oxidase (ACO) has been desired for the determination of free fatty acids (FFAs). In order to meet this demand, we prepared recombinant ACO from Arthrobacter ureafaciens NBRC 12140. The coding region of the gene was 2109, encoding a protein of 703 amino acids with a predicted molecular mass of 76.5 kDa. The heterologous expression level in Escherichia coli was 520-fold higher than that in the native strain. The purified enzyme retained more than 60% activity after incubation in the presence of 10 mM NEM at 37 degrees C for 4 h, while other commercially available ACOs showed only less than 10% activities after the same NEM treatment. We presume that this is due to the presence of only three cysteines in ACO from A. ureafaciens. Site-directed mutagenesis studies and close scrutiny of the three-dimensional structures of other related ACOs suggested that these cysteines were buried in the protein and unreactive to NEM. The recombinant enzyme was used for the colorimetric determination of free fatty acid, which gave a linear calibration.     

Rho-kinase modulates the function of STEF, a Rac GEF, through its phosphorylation

Rho family GTPases are key regulators of various physiological processes. Several recent studies indicated that the antagonistic relationship between Rho and Rac is essential for cell polarity and that the Rac activity is negatively regulated by Rho. In this study, we found that Rho-kinase, an effector of Rho, counteracted the Rac GEF STEF-induced Rac1 activation in COS7 cells. Rho-kinase phosphorylated STEF at Thr1662 in vitro, and Y-27632, a Rho-kinase inhibitor, suppressed lysophosphatidic acid-induced phosphorylation of STEF in PC12D cells. STEF interacted with specific molecules such as microtubule-associated protein 1B, and the phosphorylation of STEF by Rho-kinase diminished its interaction with these molecules. STEF promoted nerve growth factor-induced neurite outgrowth in PC12D cells, while the phosphomimic mutant of STEF had a weakened ability to enhance neurite outgrowth. Taken together, these results suggest that the phosphorylation of STEF by Rho-kinase exerts the inhibitory effect on the function of STEF.     

Central gastrin inhibits feeding behavior and food passage in neonatal chicks.

The gastrin/cholecystokinin (CCK) family is recognized as the principal family of hormones involved in regulation of the gastrointestinal tract CCK is recognized as a satiety hormone in mammalian species, but it has been suggested that gastrin rather CCK may have an important role in controlling feeding behavior in the neonatal chick through a poorly developed blood brain barrier. So far, however, there is no direct evidence that central gastrin inhibits food intake in neonatal chicks. The aim of this study was to elucidate whether central administration of gastrin 1) inhibits feeding behavior and 2) alters food passage from the crop. The effects of central administration of gastrin on food intake were investigated in experiment 1. Birds (2-day-old) were food-deprived for 3 h and then gastrin or saline was injected intracerebroventricularly. Gastrin strongly inhibited food intake in a dose-dependent fashion for 2 h. Thereafter, the effects of central gastrin on feeding behavior and serum corticosterone concentration were examined in experiment 2. Following central administration of gastrin, food intake was depressed and pecking behavior was inhibited. Serum corticosterone concentration was not altered by central administration of gastrin. The influence of central gastrin on food passage from the crop was investigated in experiment 3. Central administration of gastrin clearly delayed food passage. In conclusion, central gastrin appears to have a strong effect for the satiety and gastrointestinal motility in the neonatal chick.