Ca2+ is Required by Clubroot Resistant Turnip Cells for Transient Increases in PAL Activity that Follow Inoculation with Plasmodiophora brassicae

Phenylalanine ammonia‐lyase (PAL, EC 4.3.1.5) activity in clubroot disease‐resistant turnip calli was transiently increased by 20 h after the inoculation with Plasmodiophora brassicae spores. The magnitude of the increase in PAL activity was four to six times higher than constitutive PAL activity. There was no transient increase in PAL activity in susceptible calli. Preincubation of calli in Ca²⁺‐free medium or the removal of Ca²⁺ from cell surfaces by ethylene glycol bis(2‐aminoethyl ether)‐N,N,N′,N′‐tetraacetic acid‐chelation, completely inhibited induced PAL activity. The influx of exogenous Ca²⁺ into cells appears necessary for this pathogen induced PAL activity. Verapamil and the calmodulin inhibitor W7 almost completely inhibited induced PAL activity at 1 and 0.1 mm , respectively. Neomycin, ruthenium red and (1‐(6‐[(17β‐3‐Methoxyestra‐1,3,5‐(10)‐trien‐17‐yl)amino]hexyl)‐1H‐pyrrole‐2,5‐dione) did not inhibit induced PAL activity. Thus, verapamil and N‐(6‐aminohexyl)‐5‐chloro‐1‐naphthalenesulphonamide hydrochloride‐sensitive Ca²⁺‐mediated signalling process appear necessary for P. brassicae induced PAL activity. As the protein synthesis inhibitor cycloheximide (CHX) blocked the induced increasing PAL activity, de novo synthesis of PAL appears to be required for turnip cell defence reactions against P. brassicae.     

Identification of 4-Chloroindole-3-acetic Acid and Its Methyl Ester in Immature Seeds of Vicia amurensis (the Tribe Vicieae), and Their Absence from Three Species of Phaseoleae

The natural chlorinated auxins 4-chloroindole-3-acetic acid(4-Cl-IAA) and its methyl ester (4-Cl-IAA Me ester) were found,in addition to IAA and its Me ester, by gas chromatography-massspectrometry in immature seeds of Vicia amurensis, a Vicieaespecies. In contrast, only non-chlorinated, IAA and IAA Me esterwere present in immature seeds of three Phaseoleae species.These results are further evidence of the wide distributionof 4-Cl-IAA and its Me ester in various Vicieae. (Received October 3, 1986; Accepted December 22, 1986)     

Characterization of a novel acidic protein of 38 kDa, A0, in yeast ribosomes which immunologically cross-reacts with the 13 kDa acidic ribosomal proteins, A1/A2

A new ribosomal protein of 38 kDa, named A0, was detected in yeast ribosomes on immunoblotting. The antibody used here was that against A1/A2, 13 kDa acidic ribosomal proteins which cross-reacted with A0. Although A0 and A1/A2 share common antigenic determinants, they differ in the following biochemical properties. While A1/A2 could be extracted from ribosomes with ethanol and ammonium sulfate, A0 could not. A0 gave two protein spots in a less acidic region than for A1/A2 on two-dimensional gel electrophoresis. The heterogeneity observed for A0 was ascribable to phosphorylation because one spot disappeared after treatment of the ribosomes with phosphatase. The syntheses of A0 and A1/A2 are directed by different mRNA species, as judged with a cell-free translation system, ruling out the possibility that A0 is a precursor of A1/A2. Although a mammalian ribosomal protein equivalent to A0 has been shown to be associated with 13 kDa acidic proteins in the cytoplasm, essentially no A0 was detected on immunoblotting in the yeast cytosol, while a small but detectable amount of A1/A2 was present. The possibility that A0 is a eukaryotic equivalent of L10 of Escherichia coli is discussed.     

The mechanism of action of ricin and related toxic lectins on eukaryotic ribosomes. The site and the characteristics of the modification in 28 S ribosomal RNA caused by the toxins

Ricin is a potent cytotoxic protein derived from the higher plant Ricinus communis that inactivates eukaryotic ribosomes. In this paper we have studied the mechanism of action of ricin A-chain on rat liver ribosomes in vitro. Our findings indicate that the toxin inactivates the ribosomes by modifying both or either of two nucleoside residues, G4323 and A4324, in 28 S rRNA. These nucleotides are located close to the alpha-sarcin cleavage site and become resistant to all ribonucleases tested. The examination of the lability of phosphodiester bonds of these nucleotides to both mild alkaline digestion and aniline treatment at acidic pH suggests that the base of A4324 is removed by the toxin. This unique activity of ricin A-chain was also observed when naked 28 S rRNA is used as a substrate, indicating that the toxin directly acts on the RNA. Similar activity on 28 S rRNA is also exhibited by abrin and modeccin, ricin-related toxins, suggesting a general mechanistic pathway for ribosome inactivation by lectin toxins.     

ATL-derived factor (ADF), an IL-2 receptor/Tac inducer homologous to thioredoxin; possible involvement of dithiol-reduction in the IL-2 receptor induction.

HTLV-I transformed T cells not only express a large number of interleukin-2 receptors [IL-2R/p55(Tac)], but also produce a factor named ATL-derived factor (ADF) that augments the expression of IL-2R/p55(Tac). Based on a partial N-terminal amino acid sequence, complementary DNA (cDNA) clones for human and mouse ADF were isolated and sequenced. Recombinant ADF produced by COS-7 monkey kidney cells showed IL-2R/Tac inducing activity on YT cells, which are sensitive for ADF. ADF mRNA was strongly expressed in HTLV-I(+) T cells lines, but not in inactivated cells (THP-1, unstimulated PBMC). Furthermore, in normal human peripheral blood mononuclear cells, the expression of ADF mRNA was enhanced by mitogens or phorbol myristate acetate, suggesting a possible involvement of ADF in the lymphocyte activation. Homology analysis revealed an unexpected relationship between ADF and dithiol-reducing enzyme, thioredoxin, involved in many important biological reactions such as the conversion of ribonucleotides into deoxyribonucleotides, or the stabilization of glucocorticoid receptors. The biological significance of the generation of a redox potential in lymphocyte activation, and the possible involvement of dithiol reduction in the induction of IL-2R/Tac are discussed.     

Effects of vasoactive intestinal contractor (VIC) and endothelin on intracellular calcium level in neuroblastoma NG108-15 cells

Effects on [Ca2+]i levels of endothelin-l (ET) and vasoactive intestinal contractor peptide (VIC), which is a novel member of the endothelin family, were examined in fura 2-loaded neuroblastoma NG108-15 cells. VIC was found to be a very effective stimulus for intracellular Ca2+ mobilization and to be more potent than ET. Intracellular calcium response to sequential addition of two stimulants exhibited the homologous desensitization of either ET or VIC, but no heterologous desensitization between ET and VIC. This indicates evidence suggesting that these two peptides act through distinct receptors.     

Monooxygenation of N-acetylhistamine mediated by L-ascorbate

In the presence of molecular oxygen and a catalytic amount of copper(II) ion, ascorbate almost completely degraded histamine (approx. 72%). The reaction was shown to occur at the imidazole group but not at the primary amino group in histamine. 4-[2-(Acetylamino)ethyl]-2,3-dihydroimidazol-2-one, a monooxygenated form of N-acetylhistamine, was first isolated as the primary product.     

Changes of DNA methylation level during pre-and postnatal periods in mice

DNA methylation in an adult mammalian body shows tissue-specificity. But when and how the specificity is established in the process of development has not yet been elucidated. Here we have investigated age-dependent changes in the amount of 5-methyldeoxycytidine (5mdC) that DNA of various mouse tissues contains during the late-fetal and postnatal periods, using high-performance liquid chromatography. The tissue-specificity in the 5mdC level was observed in the late-fetal stage, and the level continued to change during the subsequent periods. The most pronounced alterations were observed in brain and liver, where similar biphasic changes were seen, but at different ages. At maturation, the 5mdC levels were high in thymus, spleen and brain, intermediate in lung, and low in liver and sperm. The data demonstrate the importance of the peri- and postnatal periods in establishment of tissue-specificity in 5mdC content.     

Nutritional characteristics of a neoglycoprotein, casein modified covalently by glucose

Glucose was combined covalently with the epsilon-amino groups of lysyl residues of bovine casein in the presence of sodium cyanoborohydride as a reducing reagent by reductive alkylation, forming stable secondary amine linkages. Solubility characteristics and nutritional values of the neoglycoprotein were examined. The degree of modification (%) of the glucosylated casein was 82.5. Solubility of the modified casein was increased by the attachment of glucose. The modification did not disturb the digestion of casein by pepsin or trypsin. Rat feeding experiments using 10% protein diets demonstrated that the protein efficiency ratio (PER) of the modified casein was 0.35 +/- 0.33 compared with 2.99 +/- 0.29 for the unmodified casein. When the modified casein was supplemented with L-lysine to equal the level of total lysine of unmodified casein, the PER value was increased to 2.21 +/- 0.29. Nitrogen balance experiments showed that the modified casein was digested completely. On the other hand, biological value and net protein utilization of the modified protein were shown to be considerably lower than those of the unmodified casein.