An Arabinogalactan-protein from Rape Leaves

A l-fucose-containing arabinogalactan-protein that strongly inhibited hemagglutination by eel anti-H agglutinin of human O erythrocytes was purified from hot phosphate-buffered saline extracts of mature leaves of rape, Brassica campestris. The purified glycoconjugate consisted of 90% of the polysaccharide moiety comprising l-fucose, l-arabinose, d-galactose, 4-O-methyl-d-glucuronic acid, and d-glucuronic acid, and 4% of the hydroxyproline-rich protein portion. Upon methylation, periodate oxidation, and enzymatic degradation, we found that consecutive β-(→3)-linked d-galactopyranosyl residues constituted a backbone chain of the polysaccharide moiety, to which the side chains of β-(→6)-linked d-galactopyranosyl residues were attached through O-6. Most of l-arabinofuranosyl residues were linked as single units through 0-3 to the side chains while a small quantity of the sugar was present as (1→2)-, (1→3)-, or (1→5)-linked inter-chain residues. Single residues of α-l-fucopyranose, apparently attached to (1→2)-linked l-arabinofuranosyl residues, reacted with eel anti-H precipitin and Aleuria aurantia l-fucose-specific lectin, and were assumed to be crucial in the expression of the H-like activity. The uronosyl residues were also located at the non-reducing terminal ends. Reductive alkaline degradation of the arabinogalactan-protein provided indications that the polysaccharide chains were mainly conjugated through serine-O-glycosidic linkages to the polypeptide core. In an immunoprecipitation test, the rape leaf arabinogalactan-protein cross-reacted with antisera raised against radish leaf arabinogalactan-protein, indicating that these cruciferous arabinogalactan-proteins share common immunodeterminant(s) in their molecules.     

Metabolism of [3H] Gibberellin A5 in Cell Suspension Cultures of Pharbitis nil

Gibberellin A₅ (GA₅), a native GA of immature seeds of Pharbitis nil, was fed to Pharbitis nil cell suspension cultures as [C-l, ³H] GA₅ (3.1 Ci/mmol), and its metabolism over a 48 hr period was investigated. Radioactivity in free GA metabolites was 13.1%, with 79.9% in GA glucosyl conjugate-like metabolites. Only 7.0% of the radioactivity remained as [³H] GA₅. Tentative identifications were based on comparison with retention times of authentic free GAs and/or glucosyl conjugates after sequential chromatography on Si gel partition column → gradient-eluted C18 HPLC-radiocounting (RC) → isocratic-eluted C18 HPLC-RC, and showed that [³H] GA₅ was converted to [³H] GA₁ (2%), [³H] GA₃ (4%), [³H] GA₆ (2%), [³H] GA₂₂ (1%) and their glucosyl conjugates, and also to [³H] GA₈ glucoside, and [³H] GA₅ glucosyl conjugates. The major conjugate-like substances were [³H] GA₁ and [³H] GA₃ glucosyl esters, at 15% and 34%, respectively, of the total extractable radioactivity.     

Production of L-Threonine by Mutants Resistant to Both α-Amino-β-hydroxyvaleric Acid and S-(2-Aminoethyl)-L-cysteine Derived from Brevibacterium flavum

Growth of Brevibacterium flavum FA-1-30 and FA-3-115, L-lysine producers derived from Br. flavum No. 2247 as S-(2-aminoethyl)-L-cysteine (AEC) resistant mutants, was inhibited by α-amino-β-hydroxyvaleric acid (AHV), and this inhibition was reversed by L-threonine. All the tested AHV resistant mutants derived from FA-1-30 accumulated more than 4 g/liter of L-threonine in media containing 10% glucose, and the best producer, FAB-44, selected on a medium containing 5 mg/ml of AHV produced about 15 g/liter of L-threonine. Many of AHV resistant mutants selected on a medium containing 2 mg/ml of AHV accumulated L-lysine as well as L-threonine, AHV resistant mutants derived from FA-3-115 produced 10.7 g/liter of L-threonine maximally. AEC resistant mutants derived from strains BB–82 and BB–69, which were L-threonine producers derived from Br. flavum No. 2247 as AHV resistant mutants, did not produce L-threonine more than the parental strains, and moreover, many of them did not accumulate L-threonine but L-lysine. Homoserine dehydrogenases of crude extracts from L-threonine producing AHV resistant mutants derived from FA–1–30 and FA–3–115 were insensitive to the inhibition by L-threonine, and those of L-threonine and L-lysine producing AHV resistant mutants from FA–1–30 were partially sensitive.

Correlation between L-threonine or L-lysine production and regulations of enzymatic activities of the mutants was discussed.     

Properties of Nucleoside Phosphorylase from Enterobacter aerogenes

The properties of uridine Phosphorylase (UPase) and purine nucleoside Phosphorylase (PNPase) at high temperature were investigated. Both enzymes were found to be distributed in a wide range of bacteria and were partially purified from Enterobacter aerogenes AJ 11125 by heat treatment, ammonium sulfate fractionation and column chromatographies onDEAE-cellulose and Sephadex G-150. The UPase was purified 109-fold, and it showed an optimum pH of 8.5 and optimum temperature of 65°C, and activity toward uridine, 2′-deoxyuridine, thymidine and uracil arabinoside but not cytidine. The Km values of UPase for uridine were 0.7 mm at 40°C and 1.8 mm at 60°C. The PNPase was purified 83-fold, and it showed an optimum pH of 6.8 and optimum temperature of 60°C, and significant activity toward purine arabinosides as well as purine ribosides. The Km values of PNPase for inosine were 0.8 mm at 40°C and 2.2 mm at 60°C.     

An Evidence for the Repair of Double-Strand Scissions in DNA of Micrococcus radiodurans after Alpha Bombardment

The conversion of prochaetoglobosins as plausible precursors into mycotoxin chaetoglobosin A (1) in a cell-free system of Chaetomium subaffine was unsuccessful. However, reductase activity of the 20-keto-analogues (1), and prochaetoglobosins II (5) and III (6) were found in a microsomal fraction of this fungi. Two new metabolites of chaetoglobosins, named chaetoglobosin F_ex (2) and 20-dihydro-chaetoglobosin A (3), were also isolated from the same micro-organisms. Their structures were elucidated by spectroscopic data and chemical transformation.     

Minor Constituents of Japanese Ho-Leaf Oil

The main component of Japanese Ho-leaf oil has been shown to be (?)-linalool (80~90%), and the following twenty minor constituents newly have been identified; methyl vinyl ketone, methyl isobutyl ketone, mesityl oxide, β-pinene, myrcene, (+)-limonene, cis- and trans-ocimene, n-hexanol, cis-3-hexenol, cis- and trans-linalool oxide, (?)-1-terpinen-4-ol, (+)-cis and (+)-trans-2,6,6-trimethyl-2-vinyl-5-hydroxytetrahydropyran, citronellol, nerol, (+)-β-selinene, (+)-tagetonol and (?)-trans-hotrienol. (+)-Tagetonol and (?)-trans-hotrienol have been demonstrated to be (+)-3,7-dimethyl-3-hydroxy-1-octen-5-one (III) and (3R)-(?)-trans-3,7-dimethyl-3-hydroxy-1,5,7-octatriene (IX), respectively.     

Fermentative Production of l-Leucine with Auxotrophic Mutants of Corynebaeterium glutamicum

An auxotrophic mutant of Corynebaeterium glutamicum was found to accumulate a large amount of l-leucine in the culture medium. The nutritional requirement of the mutant is rather complex but it’s growth was most remarkably stimulated by l-phenylalanine. Acetate (1.5~3.0%) or pyruvate (3%) stimulated the l-leucine production. By a further mutagenic treatment, 329 mutants earring some defect in addition to phenylalanine auxotrophy were derived from the mutant No. 190. Among them, a histidine auxotrophic derivative produced twice as much l-leucine as the parent strain, i.e., the level of l-leucine production by this derivative reached 16 mg/ml in a medium containing 12% glucose, 1 % (NH₄)₂SO₄ and 2.5% CH₃COONH₄ as carbon and nitrogen sources. Some other auxotrophic markers such as isoleucine- (or threonine-), threonine-, purine(s)-, homoserine-, or methionine- auxotrophy also improved the L-leucine production by No, 190.     

Studies on Biologically Active Substances Formed by Bacilli

The products of several Bacillus strains were investigated on rabbit serum calcium decreasing, oxytocic and toad heart function promoting activities. These products were obtained from the clear supernatant fluid of the culture medium after the cells were removed by centrifugation.

For the production of rabbit serum calcium decreasing substance, Bacillus subtilis K and Bacillus natto No. 8 were found to be usefull, Bacillus megaterium KM, Bacillus cereus var. mycoides and Bacillus subtilis K produced oxytocic principle. Bacillus subtilis K, Bacillus brevis and Bacillus megaterium KM also produced toad heart function promoting factor.

A procedure was developed to obtain the electrophoretically homogenous rabbit serum calcium decreasing substance from culture filtrate of Bacillus subtilis K. The crude substance was obtained as isoelectric precipitate by adjusting the culture filtrate to pH 3.0. The crude substance was purified by gel filtration on a Sephadex G-75 column, isoelectric fractionation and chromatography on DEAE-cellulose column. The purified preparation was shown to be homogenous by Tiselius electrophoresis but was separated into two bands by polyacrylamide electrophoresis. The chemical analysis of this biologically active substance indicated this substance to be a lipoprotein. The substance decreased rabbit serum calcium level about 12% at 6~8hr after intravenous injection (dose; 0.5 mg/kg body weight).     

Impact of Histone H1 on the Progression of Allergic Rhinitis and Its Suppression by Neutralizing Antibody in Mice

Nuclear antigens are known to trigger off innate and adaptive immune responses. Recent studies have found that the complex of nucleic acids and core histones that are derived from damaged cells may regulate allergic responses. However, no fundamental study has been performed concerning the role of linker histone H1 in mast cell-mediated type I hyperreactivity. In this study, we explored the impact of histone H1 on mast cell-mediated allergic responses both in vitro and in vivo. In the course of a bona-fide experimental allergen sensitization model upon co-injection with alum adjuvant, ovalbumin (OVA), but not PBS, induced elevated levels of circulating histone H1. Intranasal challenge with histone H1 to OVA/alum- (but not PBS/alum)-sensitized mice induced significantly severer symptoms of allergic rhinitis than those in mice sensitized and challenged with OVA. A monoclonal antibody against histone H1 not only suppressed mast cell degranulation, but also ameliorated OVA-induced nasal hyperreactivity and IgE-mediated passive cutaneous anaphylaxis. Our present data suggest that nuclear histone H1 represents an alarmin-like endogenous mediator acting on mast cells, and that its blockage has a therapeutic potential for mast cell-mediated type I hyperreactivity.