Habituation in suspension-cultured soybean cells to thiamine and its precursors

When thiamine concentration in subculture medium was rapidlylowered to nil, soybean cells in suspension became necroticand stopped growing entirely. When it was gradually lowered,cell growth was vigorous until the concentration was reducedto 7.8?10^–3 mg/liter. The cells at this level of thiamineceased growing for a time, but prolonged culture in the samemedium resulted in the appearance of fresh white cells whichcould be easily distinguished from the old brown, necrotic cellsin the aggregates. These new cell lines could be subculturedwith further reduction in the thiamine supply, growing as largeraggregates of about 4 mm in diameter. New cell lines were similarly obtained by prolonged culturesin media containing a thiamine precursor; three lines appearedto be habituated to the pyrimidine moiety and one to the thiazolemoiety. The latter cell line could be subcultured without thiamineand its precursors for at least eight passages. These habituatedcells were characterized by the increase of the dry to freshweight ratio and by their growth in large aggregates. ¹Present address: Section of Phytochemical Research, Eisai Co.,Ltd., Kawashima, Gifu 483, Japan. (Received December 15, 1978; )     

A self-organizing neural network model for the development of complex cells

On the basis of recent neurophysiological findings on the mammalian visual cortex, a selforganizing neural network model is proposed for the understanding of the development of complex cells. The model is composed of two kinds of connections from LGN cells to a complex cell. One is direct excitatory connections and the other is indirect inhibitory connections via simple cells. Inhibitory synapses between simple cells and complex cells are assumed to be modifiable. The model was simulated on a computer to confirm its behavior.     

Stereoselective hydrolysis of dl-menthyl succinate by gel-entrapped Rhodotorula minuta var. texensis cells in organic solvent

Summary dl-Menthyl succinate was successfully hydrolyzed stereoselectively by Rhodotorula minuta var. texensis cells entrapped within photo-crosslinked or polyurethane resin gels in water-saturated n-heptane. The hydrolyzed product was found to be pure l-menthol. The catalytic activity of the immobilized cells, especially those entrapped in urethane polymers, was far more stable than that of the free cells. The half-life of the polyurethaneentrapped cells was estimated to be 55–63 days in the organic solvent.Dedicated to the 65th birthday od Professor Dr. G. Manecke     

Partial purification and property of pyridoxine (pyridoxamine)-5′-phosphate oxidase isozymes from wheat seedlings

Isozymes of pyridoxine (pyridoxamine)-5′-phosphate oxidase (EC 1.4.3.5) were isolated from the extract of wheat seedlings by column chromatographies. From DEAE-Sephadex A-50, two fractions having pyridoxine-5′-phosphate oxidase activity were separated by eluting with ~0.075 and ~0.125 m phosphate buffers (pH 8.0). These fractions were further fractionated on a Blue-Sepharose CL-6B column, from which again two activities were eluted by 1.0 m KCl solution. One fraction, designated as E-I, used only pyridoxine 5′-phosphate as substrate, whereas the other, designated as E-II, oxidized not only pyridoxine 5′-phosphate but also pyridoxamine 5′-phosphate with approximately equal rates. The mobility on polyacrylamide disc gel electrophoresis and the substrate specificity of these two fractions were different. Therefore, they were concluded to be isozymes.     

A cytolysin, theta-toxin, preferentially binds to membrane cholesterol surrounded by phospholipids with 18-carbon hydrocarbon chains in cholesterol-rich region.

We have previously suggested the existence of two distinctive states of cholesterol in erythrocyte and lymphoma cell membranes as revealed by high- and low-affinity binding sites for theta-toxin of Clostridium perfringens [Ohno-Iwashita, Y., Iwamoto, M., Mitsui, K., Ando, S., & Nagai, Y. (1988) Eur. J. Biochem. 176, 95-101; Ohno-Iwashita, Y., Iwamoto, M., Ando, S., Mitsui, K., & Iwashita, S. (1990) Biochim. Biophys. Acta 1023, 441-448]. To understand factor(s) which determine membrane cholesterol heterogeneity, we analyzed toxin binding to large unilamellar liposomes composed of cholesterol and phospholipids (phosphatidylcholine/phosphatidylglycerol = 82:18, mol/mol). Liposomes containing phospholipids with 18-carbon hydrocarbon chains at both positions 1 and 2 of the glycerol have both high- and low-affinity toxin-binding sites with Kd values similar to those of intact erythrocytes, whereas liposomes with hydrocarbon chains containing 16 or fewer carbons at either position 1 or 2 have only low-affinity toxin-binding sites. The cholesterol/phospholipid ratio, in addition to the length of phospholipid hydrocarbon chain, also determines the number of toxin-binding sites, indicating that at least these two factors determine the topology of membrane cholesterol by creating distinctively different affinity sites for the toxin. Since theta-toxin binding detects specific populations of membrane cholesterol that are not detectable by the measurements of susceptibility to cholesterol oxidase and cholesterol desorption from membranes, the toxin could provide a unique probe for studying the organization of cholesterol in membranes.     

Two genes, atpC1 and atpC2, for the gamma subunit of Arabidopsis thaliana chloroplast ATP synthase.

Arabidopsis thaliana has two genes (atpC1, atpC2) coding for gamma subunits of chloroplast ATP synthase. The atpC1 and atpC2 were cloned and sequenced. They had no introns within the reading frames and coded for proteins of 373 and 386 amino acid residues, respectively, including putative transit sequences (50 and 60 amino acid residues, respectively). In contrast, the spinach gamma subunit gene had two introns within the reading frame. The mature sequences coded by the two genes of A. thaliana (atpC1, 323 residues; atpC2, 326 residues) were homologous with that of spinach (J. Miki, M. Maeda, Y. Mukohata, and M. Futai (1988) FEBS Lett. 232, 221-226): the homologies of gamma subunits coded by atpC1 and atpC2 were 72%, those of the subunits coded by atpC1 and spinach cDNA were 84%, and those of the proteins coded by atpC2 and spinach cDNA were 71%. Like the spinach subunit, the gamma subunits coded by the two genes had unique regulatory domains not found in mitochondrial or bacterial subunits. Poly(A)+ mRNAs corresponding to atpC1 (1.5 kilobases) and atpC2 (2.5 kilobases) were detected in illuminated plants, the amount of the former being at least 140 times that of the latter. The atpC1 mRNA was not found in dark-adapted plants. Nuclear protein(s) specifically bound to the upstream region of atpC1 was detected by gel shift assay and its binding was shown to be inhibited by the GT-1 element of the gene encoding the ribulose-1,5-bisphosphate carboxylase small subunit, which is expressed under illumination (P. J. Green, S. A. Kay, and N. H. Chau (1987) EMBO J. 6, 2543-2549). Consistent with these findings, an increased amount of the gamma subunit was detected immunochemically in illuminated plants.     

One-step purification of Escherichia coli H(+)-ATPase (F0F1) and its reconstitution into liposomes with neurotransmitter transporters.

About 30% of the protein in the inner membrane of Escherichia coli strain DK8/pBWU13 is H(+)-ATPase (F0F1), and practically homogeneous F0F1 could be obtained by gradient centrifugation after solubilization of these membranes. The recombinant plasmid pBWU13 carries the unc operon for F0F1. When reconstituted into liposomes, F0F1 formed an ATP-dependent proton gradient and membrane potential. Proteoliposomes reconstituted with F0F1 and solubilized transporters from chromaffin granules or synaptic vesicle membranes could transport serotonin, dopamine, and norepinephrine dependent on ATP hydrolysis. F0F1 can be obtained rapidly from DK8/pBWU13, and its reconstitution into liposomes with transporters may be useful for monitoring these transporters during their purification.     

Evidence of synergy between Thy-1 and CD3/TCR complex in signal delivery to murine thymocytes for cell death.

The potential role of Thy-1 in CD3/TCR complex-mediated signal delivery to murine thymocytes was studied. Ag-mimicking cross-linked anti-CD3 mAb stimulated suspension of thymocytes from adult (6 to 8 wk old) mice for a brisk free cytoplasmic calcium ion ([Ca2+]i) rise, low level of inositol phosphate production, and marginal increase in tyrosine-specific phosphorylation of 110/120-kDa and 40-kDa cellular proteins. Weak but sustained [Ca2+]i rise, low inositol phosphate production, and weak protein tyrosine phosphorylation were also induced by the cross-linked anti-Thy-1 mAb that mimicked the putative natural ligand. The signal delivered via either of these two pathways was however insufficient for definitively promoting cell death and DNA fragmentation in the adult thymocytes. Here we demonstrated that anti-Thy-1 mAb synergized with anti-CD3 mAb for inducing a long-lasting prominent [Ca2+]i rise, definite inositol 1,4,5-triphosphate and inositol 1,3,4,5-tetrakiphosphate production, and extensive tyrosine-specific phosphorylation of 110/120-, 92-, 75-, and 40-kDa proteins, which resulted in marked promotion of cell death and DNA fragmentation in the adult thymocytes. This unique anti-Thy-1 antibody activity was confirmed to be directed to glycosylphosphatidylinositol-anchored Thy-1, and was distinguished from the known anti-L3T4 activity that augmented the CD3-mediated signal transduction in a different manner. The synergistic actions of anti-CD3 and anti-Thy-1 mAb obligatorily required the cross-linking of the two mAb together. The anti-CD3 and anti-Thy-1 mAb cross-linked together acted on immature thymocytes from newborn (less than 24 h after birth) mice for rather more extensive promotion of protein tyrosine phosphorylation and cell death. In addition, they affected peripheral T lymphocytes for accelerating protein tyrosine phosphorylation but not cell death. These results suggest a novel function of glycosylphosphatidylinositol-anchored Thy-1 as a possible unique intrathymic intensifier of the CD3/TCR complex-delivered signal for negative thymocyte selection.