Structure Elucidation of Gibberellin A32 and Its Acetonide

The structures of two gibberellin-like substances isolated from the immature seeds of Prunus persica, tentatively named PG–I and PG–II, were elucidated. PG–I was an ammonium salt of a novel gibberellin, ent-3α,10,12β,13,15α-pentahydroxy-20-norgibberella-l,16-diene-7,19-dioic-19,10-lactone (1), to which gibberellin number A₃₂ was allocated. PG–II was shown to be gibberellin A₃₂ acetonide (7), and concluded to be an artifact produced from gibberellin A₃₂ in the isolation process.     

Microbial Production of Plant Gibberellins and Related Compounds from ent-Kaurene Derivatives in Gibberella fujikuroi

ent-15α-Hydroxykaurenoic acid (8) was synthesized and fed to a mycelium suspension of Gibberella fujikuroi in the presence of 1-n-decylimidazole, a gibberellin biosynthesis inhibitor. The metabolites included 15β-hydroxy GA₂₄, GA₄₅ (GA of Pyrus communis), 15β-hydroxy GA₁₅ and 15β-hydroxy GA₂₅. Microbial production of 12α-hydroxy GAs from ent-12β-hydroxykaurene is also described.     

Gibberellins in Immature Seeds of Pharbitis nil

A new gibberellin, gibberellin A₂₀ (GA₂₀), was isolated from immature seeds of morning-glory (Pharbitis nil). Its structure was established as 4aα, 7α-dihydroxy-1β-methyl-8-methylenegibbane-1α, 10β-dicarboxylic acid-1→4a lactone (I) on the basis of its physicochemical analysis as well as chemical evidences. GA₂₀ shows marked growth promoting activities on dwarf maize d₂ and d₅ but weak activities on d₁, rice seedling and dwarf pea.     

Structures of Gibberellins A39, A48, A49 and a New Kaurenolide in Cucurbita pepo L.

The structures of three new gibberellins A₃₀, A₄₈ and A₄₉ and a new kaurenolide, isolated from seeds of Cucurbita pepo L., were elucidated. The structures of GA₃₉, GA₄₈ and GA₄₉ were shown to be ent-3α,12β-dihydroxygibberell-16-ene-7,19,20-trioic acid (1), ent-2α,3α,10,12α-tetrahydroxy-20-norgibberell-16-ene-7,19-dioic acid 19,10-lactone (5) and the epimer at C–12 of GA₄₈ (8), respectively. The kaurenolide was shown to have the structure: ent-6β,7α,12β-trihydroxykaur-16-en-19-oic acid 19,6-lactone (14).     

Thermal Stability of Individual Molecular Species of Soybean β-Conglycinin

The thermal stability of three individual molecular species of soybean β-conglycinin, α₃, β₃, and modified α₃ (amino terminal Vall-Argl26 amino acid groups deleted), was studied by the change in far- and near-ultraviolet CD spectra and in surface hydrophobicity. The α₃ molecule was less thermostable than β₃, its lower stability being derived from the core of the molecule rather than the extra polypeptide chain on the amino terminal side.     

Metabolism of Steviol and Its Derivatives by Gibberella fujikuroi

Steviol (ent-13-hydroxykaur-16-en-19-oic acid)* is metabolized by Gibberella fujikuroi in the presence of inhibitors of gibberellin biosynthesis, such as quaternary ammonium salt-type growth retardants, to afford 7β-Miydroxy- and 6β,7β-dihydroxysteviol, gibberelhns A₁, A₁₈, A₁₉, A₅₃ and 7β,13-dihydroxykaurenolide. Steviol acetate (ent-13-acetoxykaur-16-en-19-oic acid) is also metabolized to the 6β,7β-dihydroxy-derivative and to the 13-acetyl derivatives of gibberellins A₁₇ and A₂₀ and steviol methyl ester (methyl ent-13-hydroxykaur-16-en-19-oate) into the monohydroxy-, dihydroxy- and hydroxyoxo-derivatives. These results indicate a low substrate specificity of the enzymes in the fungus and provide a useful preparative methodology of several important plant gibberellins carrying the 13-hydroxyl group.     

Gibberellins in Immature Seeds of Canavalia

Two novel gibberellins, GA₂₁ (I) and GA₂₂ (II), were isolated from immature seeds of sword bean, Canavalia gladiata DC. The isolation procedure of these substances as well as their growth-promoting effects on dwarf maize mutants d₁ and d₅, rice, cucumber and dwarf peas (Progress No. 9) are described.

The structures of two new gibberellins, GA₂₁ and GA₂₂, isolated from immature seeds of sword bean, were determined as 4a_α, 7_α-dihydroxy-8-methylenegibbane-1, 1, 10β-tricarboxylic acid 1→4a lactone (II) and 4a_α, 7_α-dihydroxy-l β-hydroxymethyl-8-methylenegibb-2-ene-1_α, 10β-dicaboxylic acid 1→4a lactone (IV), respectively, on the basis of chemical and physicochemical studies.     

The identification of gibberellins in immature seeds of Vicia faba,and some chemotaxonomic considerations

GA₁₇, GA₁₉, GA₂₀, GA₂₉, GA₄₄ and 13-hydroxy-GA₁₂, now named GA₅₃, were identified by GC-MS in immature seeds of Vicia faba (broad bean). Also identified were a GA catabolite, two polyhydroxykauranoic acids, and abscisic, phaseic and dihydrophaseic acids. The GAs of Vicia are hydroxylated at C-13, in common with those of other legumes. However the GAs of Vicia are not hydroxylated at C-3, nor do they appear to be readily conjugated. In these respects Vicia resembles Pisum, another member of the tribe Viciae. Vicia differs from Phaseolus and Vigna, of the tribe Phaseoleae, in both these respects.Abbreviations ABA abscisic acid - DPA dihydrophaseic acid - GA_n gibberellin A_n - GC gas chromatography - GC-MS gas chromatography mass spectrometry - KA kauranoic acid - PA phaseic acid - TLC thin layer chromatography     

Studies on Conjugated Lipids

It has been confirmed that gibberellin A is a mixture of three components, gibberellin A₁, gibberellin A₂ and gibberellic acid (namely gibberellin X), by treating their methyl ester through the chromatography on A1₂O₃ column. Attempts to separate them in free acid were made. The physical and chemical properties of each gibberellin as well as its physiological properties are described     

The microbiological transformation of some trachylobane diterpenoids by Gibberella fujikuroi

The microbiological transformation of ent-trachylobane, ent-7α-hydroxytrachylobane and ent-19-hydroxytrachylobane into trachylobagibberellins A₇, A₉, A₁₃, A₂₅, A₄₀ and A₄₇ by Gibberella fujikuroi is described. Whereas 7β-hydroxy- and 7β,18-dihydroxytrachylobanolides were obtained from ent-trachylobane and ent-trachyloban- 19-ol, the presence of a 7β-hydroxyl group directed metabolism exclusively into the gibberellin pathway. An 18-hydroxyl group as in ent-7α,18-dihydroxytrachylobane inhibited oxidation at C-6 affording ent-7α,18,19-trihydroxytrachylobane as the major metabolite.     

Isolation of Taka-amylase A Peptides Required for Substrate Binding

An α-amylase from Aspergillus oryzae, Taka-amylase A (TAA), was cleaved into peptide fragments by an acid protease. Inactivation of TAA was greatly retarded by the addition of α-cyclodextrin or Ca²⁺. TAA peptide fragments were separated into two groups having no and high affinity to the substrate, soluble starch. This separation was done by the forced affinity chromatography method by a column of epichlorohydrin cross-linked soluble starch gel. Three peptides were isolated from the high-affinity fragments, purified by the ODS-120T column, and their amino acids were sequenced. Peptides I, II, and III originated from α2-helix, α3-helix, and β2-sheet, respectively, and all of these were located in the (β/α)₈ barrel of the main domain of TAA molecule. A stereo graphic view showed that Peptides I–III were at the cleft near the catalytic site. Occurrence of a Trp residue in all three peptides strongly suggested that Trp was very important in the binding of TAA to the substrate, soluble starch.     

5α‐Androst‐16‐en‐3α‐ol β‐D‐Glucuronide,Precursor of 5α‐Androst‐16‐en‐3α‐ol in Human Sweat

5α‐Androst‐16‐en‐3α‐ol (α‐androstenol) is an important contributor to human axilla sweat odor. It is assumed that α‐andostenol is excreted from the apocrine glands via a H₂O‐soluble conjugate, and this precursor was formally characterized in this study for the first time in human sweat. The possible H₂O‐soluble precursors, sulfate and glucuronide derivatives, were synthesized as analytical standards, i.e., α‐androstenol, β‐androstenol sulfates, 5α‐androsta‐5,16‐dien‐3β‐ol (β‐androstadienol) sulfate, α‐androstenol β‐glucuronide, α‐androstenol α‐glucuronide, β‐androstadienol β‐glucuronide, and α‐androstenol β‐glucuronide furanose. The occurrence of α‐androstenol β‐glucuronide was established by ultra performance liquid chromatography (UPLC)/MS (heated electrospray ionization (HESI)) in negative‐ion mode in pooled human sweat, containing eccrine and apocrine secretions and collected from 25 female and 24 male underarms. Its concentration was of 79 ng/ml in female secretions and 241 ng/ml in male secretions. The release of α‐androstenol was observed after incubation of the sterile human sweat or α‐androstenol β‐glucuronide with a commercial glucuronidase enzyme, the urine‐isolated bacteria Streptococcus agalactiae, and the skin bacteria Staphylococcus warneri DSM 20316, Staphylococcus haemolyticus DSM 20263, and Propionibacterium acnes ATCC 6919, reported to have β‐glucuronidase activities. We demonstrated that if α‐ and β‐androstenols and androstadienol sulfates were present in human sweat, their concentrations would be too low to be considered as potential precursors of malodors; therefore, the H₂O‐soluble precursor of α‐androstenol in apocrine secretion should be a β‐glucuronide.     

Isolation and Identification of Lipase Activators from Saccharomycopsis lipolytica

Three types of lipase activators (α, β, γ) were isolated from the culture broth of Saccharomycopsis lipolytica using high performance liquid chromatography. Activator γ was the most active for the lipase reaction. One of them (β) was identified with a mixture of 3,5-dihydro xy-7-tetradecenoic acid and related compounds by the method of NMR and GC-MS analyses. The free carboxyl group in the compounds was essential for the activation of the lipase reaction.     

Polyoxygenated ent-kauranes and water-soluble conjugates in seed of Phaseolus coccineus

C-α and C-β, previously isolated from seed of Phaseolus coccineus, are shown respectively to be the bis-O-isopropylidene and the 16,17-mono-O-isopropylidene derivatives of ent-6α,7α,16β,17-tetrahydroxykauranoic acid. By GC-MS characterization of the products of acidic, basic and enzymatic hydrolysis, water soluble conjugates of the following compounds have been shown to occur in P. coccineus seed: GA₈, GA₁₇, GA₂₀, GA₂₈, ent-6α,7α,13-trihydroxykaurenoic acid, ent-6α,7α,17-trihydroxy-16β-kauranoic acid, ent-6α,7α,16β,17-tetrahydroxykauranoic acid, 7β,13-dihydroxykaurenolide and abscisic acid.     

Conversion of [14C]gibberellin A12-aldehyde to C19- and C20-gibberellins in a cell-free system from immature seed of Phaseolus coccineus L.

A cell-free system prepared from developing seed of runner bean (Phaseolus coccineus L.) converted [¹⁴C]gibberellin A₁₂-aldehyde to several products. Thirteen of these were identified by capillary gas chromatography-mass spectrometry as gibberellin A₁ (GA₁), GA₄, GA₅, GA₆, GA₁₅, GA₁₇, GA₁₉, GA₂₀, GA₂₄, GA₃₇, GA₃₈, GA₄₄ and GA₅₃-aldehyde, all giving mass spectra with ¹⁴C-isotope peaks. GA₈ and GA₂₈ were also identified but contained no ¹⁴C. All the [¹⁴C]GA₁₂-aldehyde metabolites, except GA₁₅, GA₂₄ and GA₅₃-aldehyde, are known endogenous GAs of P. coccineus.Abbreviations GA_n gibberellin A_n - GC-MS combined gas chromatography-mass spectrometry - HPLC highperformance liquid chromatography - MVA mevalonic acid - S-2 2000-g supernatant     

A stimulatory subunit in the polypeptide elongation factor-1 of the chick brain

Abstract: The higher-molecular-weight elongation factor-1 (EF-1_H) of the chick brain was observed to contain three subunits (denominated α, β, and γ), contrary to a previous report that the brain EF-1_H consisted of aggregates of low-molecular-weight elongation factor- 1 (EF-1_L). Crude EF-1_H, obtained from 20-day embryonic brain, was treated with 0.4 M ammonium chloride and 0.1 mM GTP, and EF-1_βγ, was obtained using a DEAE-Sephadex column equilibrated in 0.025 mM GTP. Both EF-1_β, and EF-1_γ, were isolated by means of a DE-52 column equilibrated in 6 M urea and were found to have molecular weights of 2.8 and 4.8 × 10⁴, respectively. EF-1_β and EF-1_γ were also obtained from young rat and calf brains by the same procedures. The molecular weight of the isolated EF-1_α was 5 × 10⁴. It was found that EF-1_β stimulated the two EF-1_α-dependent reactions, i.e., phenylalanyl-tRNA binding (reaction 1) and polyphenylalanine synthesis (reaction 2), and also stimulated the nucleotide exchange reaction in the EF- 1_α-guanine nucleotide binary complex (reaction 3). The degrees of stimulation of reactions 1, 2, and 3 by the addition of EF-1_β were 2 to 3 times, about 18 times, and 2 to 3 times as much as with EF-1_α alone, respectively. The amino acid compositions of EF-1_α -1_β, and -1_γ and EF-2 were very similar to those of other eukaryotic tissues. Thus the constituents and properties of EFs of the brain were found to be basically similar to those of other tissues of eukaryotes, although EF-1_β, and EF-1, had not been reported in the brain. A possible physiological significance of EF-1_β during brain development is also discussed.     

Three Novel Gibberellins Produced by Gibberella fujikuroi

Three novel gibberellins, GA₅₄ (ent-1α, 3α, 10-trihydroxy-20-norgibberell-16-ene-7, 19-dioic acid 19, 10-lactone), GA₅₅ (ent-1α, 3α, 10, 13-tetrahydroxy-20-norgibberell-16-ene-7, 19-dioic acid 19, 10-lactone) and GA₅₆ (ent-2β, 3α, 10, 13-tetrahydroxy-20-norgibberell-16-ene-7, 19-dioic acid 19, 10-lactone) were shown to occur in the culture broth of Gibberella fujikuroi. Their structures were determined mainly by mass spectrometrical comparison of the derivatives with those of authentic compounds prepared from known gibberellins.     

THE LIPID COMPOSITION OF THORACOSPHAERA HEIMII: EVIDENCE FOR INCLUSION IN THE DINOPHYCEAE1

The fatty acid, sterol and chlorophyll composition of the calcified, unicellular alga Thoracosphaera heimii (Lohmann) Kamptner are reported. The presence of 4,23,24-termethyl-5α-cholest-22E-en-3β-ol (dinosterol), 4,23,24-trimethyl-5α-cholest-22E-en-3-one (dinosterone) and the predominance of C₁₈, C₂₀ and C₂₂ unsaturated fatty acids, including the acid 18:5ω3, indicates that T. heimii is a dinoflagellate. The fatty acid: sterol ratio (1.3), is typical of dinoflagellates. The geochemical significance of dinosterone, the high relative concentration of 4-desmethyl-5α-stanols and the role of 23-methyl-5α-cholest-22E-en-3β-ol in the biosynthesis of dinosterol in T. heimii are also discussed.     

Biochemical Studies on “Bakanae” Fungus. Part XXXVI The Physiological Action of Gibberellin. VIII

The seventh leaf-sheaths of rice plants grown in solution and treated with gibberellin were analyzed to examine their changes in the activities of various enzymes during period of growth, in comparison with that of the control plants. Phosphatase, alkalipyrophosphatase, acetylesterase, maltase, β-glucosidase, α-galactosidase, β-galactosidase, amylase, urease, dipeptidase, ascorbic acid oxidase, and catalase activities were decreased in extracts of sheaths on a fresh weight basis by treatment with gibberellin. Activities of peroxidase and invertase were markedly increased.     

Rapidly directional biotransformation of tauroursodeoxycholic acid through engineered <Emphasis Type="Italic">Escherichia coli</Emphasis>

Bear bile powder is a precious medicinal material. It is characterized by high content of tauroursodeoxycholic acid (TUDCA) at a ratio of 1.0–1.5 to taurochenodeoxycholic acid (TCDCA). Here, we reported the biotransformation of tauroursodeoxycholic acid (TUDCA) through Escherichia coli engineered with a two-step mimic biosynthetic pathway of TUDCA from taurochenodeoxycholic acid (TCDCA). Two 7α-hydroxysteroid dehydrogenase (7α-HSDH) and two 7β-hydroxysteroid dehydrogenase (7β-HSDH) genes (named as α₁, α₂, β₁, and β₂) were selected and synthesized to create four pathway variants using ePathBrick. All could convert TCDCA to TUDCA and the one harboring α₁ and β₂ (pα₁β₂) showed the strongest capability. Utilizing the oxidative and reductive properties of 7α- and 7β-HSDH, an ideal balance between TUDCA and TCDCA was established by optimizing the fermentation conditions. By applying the optimal condition, E. coli containing pα₁β₂ (BL-pα₁β₂) produced up to 1.61 ± 0.13 g/L of TUDCA from 3.23 g/L of TCDCA at a ratio of 1.3 to TCDCA. This study provides a potential approach for bear bile substitute production from cheap and readily available chicken bile.