Endogenous Gibberellins from Sporophytes of Two Tree Ferns, Cibotium glaucum and Dicksonia antarctica

Gibberellin A₁ (GA₁), 3-epi-GA₁, GA₄, GA₉, 11α-hydroxyGA₁₂, 12α-hydroxyGA₁₂, GA₁₅, GA₁₇, GA₁₉, GA₂₀, GA₂₅, GA₃₇, GA₄₀, GA₅₈, GA₆₉, GA₇₀, and GA₇₁ have been identified from Kovats retention indices and full scan mass spectra by capillary GC-MS analyses of purified extracts from sporophytes of the tree fern, Cibotium glaucum. Abscisic acid, dihydrophaseic acid, an epimer of 4′-dihydrophaseic acid, and the epimeric ent-6α, 7α, 16α, 17-(OH)₄ and ent-6α, 7α, 16β, 17-(OH)₄ derivatives of ent16, 17-dihydrokaurenoic acid, in addition to the epimeric 16α, 17- and 16β, 17-dihydroxy-16, 17-dihydro derivatives of GA₁₂, were also identified in extracts of C. glaucum. An oxodihydrophaseic acid and a hydroxydihydrophaseic acid were also detected. In extracts of sporophytes of Dicksonia antarctica, GA₄, GA₉, 12α- and 12β-hydroxyGA₁₂, GA₁₅, GA₂₅, and GA₃₇ were identified by the same criteria, as well as abscisic acid, phaseic acid, 8′-hydroxymethylabscisic acid and dihydrophaseic acid. This is the first time that GA₄₀ has been identified in a higher plant; it is also the first report of the natural occurrence of the two gibberellins, 11α- and 12β-hydroxyGA₁₂. The total gibberellin (GA) content in C. glaucum (tall) was at least one order of magnitude greater than that of D. antarctica (dwarf) based on total ion current response in GC-MS and bioassay data. Abscisic acid was a major component of D. antarctica and the oxodihydrophaseic acid was a major component of C. glaucum.     

Identification of endogenous gibberellins from oilseed rape

Oilseed rape (Brassica napus, canola variety `Westar') plants were grown in greenhouse conditions and shoots were harvested during the final stages of shoot elongation. Leaves and immature pods were removed and the remaining stem tissue was extracted and purified. The extract was chromatographed on sequential, step-eluted silica gel partition and reverse-phase C<sub>18</sub> HPLC columns, and gibberellin (GA)-like substances were detected using the `Tan-ginbozu' dwarf rice microdrop assay. Purified fractions showing GA-like activity were analyzed by capillary gas chromatography-mass spectrometry (GC-MS) and GC-selected ion monitoring (GC-SIM). Gibberellins A₁, A₃, and iso-A₃ were identified by full spectrum GC-MS with GA₁ being the most abundant GA in the stem tissue. Gibberellins A₁₉ and A₂₀ were identified by GC-SIM and are logical precursors of the GA₁.     

Identification of endogenous gibberellins from sorghum

Gibberellins (GA) A₁, A₁₉, and A₂₀ were identified in shoot cylinders containing the apical meristems from sorghum (Sorghum bicolor L.). Extracts were purified by sequential SiO₂ partition chromatography and reversed-phase C₁₈ high performance liquid chromatography and biologically active (dwarf rice cv Tan-ginbozu microdrop assay) fractions were subjected to gas chromatography-selected ion monitoring. Based on the use of [³H]GA and [²H](d₂)GA internal standards, amounts of GA₁, GA₁₉, and GA₂₀ were estimated to be 0.7, 8.8, and 1.5 namograms per gram dry weight of tissue, respectively.     

The metabolism of steviol to 13-hydroxylated ent-Gibberellanes and ent-kauranes

Steviol(ent-13-hydroxykaur-16-en-19-oic acid) is rapidly metabolised by the mutant B1-41a of Gibberellafujikuroi. The initial product is the ent- 7-α-hydroxy derivative which is then further metabolised to gibberellins A₁, A₁₈, A₁₉, A₂₀, 13-hydroxy GA₁₂, the ent-6α, 7α, 13- and ent-6β, 7α, 13 (19,6-lactone)-trihydroxykaurenoic acids, and a seco-ring B diacid. This apparently low substrate specificity of the enzymes operative beyond the block in the mutant B1-41a provides a useful model for the biosynthetic pathways to 13-hydroxylated gibberellins of higher plants and a preparative route to these plant gibberellins.     

Molecular probes for the A2A adenosine receptor based on a pyrazolo[4,3-e][1,2,4]triazolo[1,5-c]pyrimidin-5-amine scaffold

Pyrazolo[4,3-e][1,2,4]triazolo[1,5-c]pyrimidin-5-amine derivatives such as SCH 442416 display high affinity and selectivity as antagonists for the human A_2A adenosine receptor (AR). We extended ether-linked chain substituents at the p-position of the phenyl group using optimized O-alkylation. The conjugates included an ester, carboxylic acid and amines (for amide condensation), an alkyne (for click chemistry), a fluoropropyl group (for ¹⁸F incorporation), and fluorophore reporter groups (e.g., BODIPY conjugate 14, K_i 15 nM). The potent and A_2AAR-selective N-aminoethylacetamide 7 and N-[2-(2-aminoethyl)-aminoethyl]acetamide 8 congeners were coupled to polyamidoamine (PAMAM) G3.5 dendrimers, and the multivalent conjugates displayed high A_2AAR affinity. Theoretical docking of an AlexaFluor conjugate to the receptor X-ray structure highlighted the key interactions between the heterocyclic core and the binding pocket of the A_2AAR as well as the distal anchoring of the fluorophore. In conclusion, we have synthesized a family of high affinity functionalized congeners as pharmacological probes for studying the A_2AAR.     

p16INK4A Participates in a G1 Arrest Checkpoint in Response to DNA Damage

Members of the INK4 protein family specifically inhibit cyclin-dependent kinase 4 (cdk4) and cdk6-mediated phosphorylation of the retinoblastoma susceptibility gene product (Rb). p16^INK4A, a prototypic INK4 protein, has been identified as a tumor suppressor in many human cancers. Inactivation of p16^INK4A in tumors expressing wild-type Rb is thought to be required in order for many malignant cell types to enter S phase efficiently or to escape senescence. Here, we demonstrate another mechanism of tumor suppression by implicating p16^INK4A in a G₁ arrest checkpoint in response to DNA damage. Calu-1 non-small cell lung cancer cells, which retain Rb and lack p53, do not arrest in G₁ following DNA damage. However, engineered expression of p16^INK4A at levels compatible with cell proliferation restores a G₁ arrest checkpoint in response to treatment with γ-irradiation, topoisomerase I and II inhibitors, and cisplatin. A similar checkpoint can be demonstrated in p53^−/− fibroblasts that express p16^INK4A. DNA damage-induced G₁ arrest, which requires the expression of pocket proteins such as Rb, can be abrogated by overexpression of cdk4, kinase-inactive cdk4 variants capable of sequestering p16^INK4A, or a cdk4 variant incapable of binding p16^INK4A. After exposure to DNA-damaging agents, there was no change either in overall levels of p16^INK4A or in amounts of p16^INK4A found in complex with cdks 4 and 6. Nonetheless, p16^INK4A expression is required for the reduction in cdk4- and cdk6-mediated Rb kinase activity observed in response to DNA damage. During tumor progression, loss of p16^INK4A expression may be necessary for cells with wild-type Rb to bypass this G₁ arrest checkpoint and attain a fully transformed phenotype.     

Comparison of Biological Activities of Gibberellins and Gibberellin-Precursors Native to Thlaspi arvense L

Field pennycress (Thlaspi arvense L.) is a winter annual weed with a cold requirement for stem elongation and flowering. The relative abilities of several native gibberellins (GAs) and GA-precursors to elicit stem growth were compared. Of the eight compounds tested, gibberellin A₁, (GA₁), GA₉, and GA₂₀ caused stem growth in noninduced (no cold treatment) plants. No stem growth was observed in plants treated with ent-kaurene, ent-kaurenol, ent-kaurenoic acid, GA₅₃, or GA₈. Moreover, of the biologically active compounds, GA₉ was the most active followed closely by GA₁. In thermoinduced plants (4-week cold treatment at 6°C) that were continuously treated with 2-chlorocholine chloride to reduce endogenous GA production, GA₉ was the most biologically active compound. However, the three kaurenoid GA precursors also promoted stem growth in thermoinduced plants, and were almost as active as GA₂₀. No such increase in activity was observed for either GA_[unk] or GA₅₃. The results are discussed in relation to thermoinductive regulation of GA metabolism and its significance to the initiation of stem growth in field pennycress. It is proposed that thermoinduction results in increased conversion of ent-kaurenoic acid to GAs through the C-13 desoxy pathway and that GA₉ is the endogenous mediator of thermoinduced stem growth in field pennycress.     

Internode Length in Pisum: Gene na May Block Gibberellin Synthesis between ent-7alpha-Hydroxykaurenoic Acid and Gibberellin A(12)-Aldehyde

The elongation response of the gibberellin (GA) deficient genotypes na, ls, and lh of peas (Pisum sativum L.) to a range of GA-precursors was examined. Plants possessing gene na did not respond to precursors in the GA biosynthetic pathway prior to GA₁₂-aldehyde. In contrast, plants possessing lh and ls responded as well as wild-type plants (dwarfed with AMO-1618) to these compounds. The results suggest that GA biosynthesis is blocked prior to ent-kaurene in the lh and ls mutants and between ent-7α-hydroxykaurenoic acid and GA₁₂-aldehyde in the na mutant. Feeds of ent-[³H]kaurenoic acid and [²H]GA₁₂-aldehyde to a range of genotypes supported the above conclusions. The na line WL1766 was shown by gas chromatography-mass spectrometry (GC-MS) to metabolize [²H]GA₁₂-aldehyde to a number of[²H]C₁₉-GAs including GA₁. However, there was no indication in na genotypes for the metabolism of ent-[³H]kaurenoic acid to these GAs. In contrast, the expanding shoot tissue of all Na genotypes examined metabolised ent-[³H]kaurenoic acid to radioactive compounds that co-chromatographed with GA₁, GA₈, GA₂₀, and GA₂₉. However, insufficient material was present for unequivocal identification of the metabolites. The radioactive profiles from HPLC of extracts of the node treated with ent-[³H]kaurenoic acid were similar for both Na and na plants and contained ent-16α,17-dihydroxykaurenoic acid and ent-6α,7α,16β,17-tetrahydroxykaurenoic acid (both characterized by GC-MS), suggesting that the metabolites arose from side branches of the main GA-biosynthetic pathway. Thus, both Na and na plants appear capable of ent-7α-hydroxylation.     

Studies on monoacylglycerols from Gordona lentifragmenta,Gordona bronchialis and Gordona rubropertincta

Monoacylglycerols containing α-branched-β-hydroxylated fatty acids (mycolic acids) ranging from C₄₂ to C₅₀ and from C₆₀ to C₆₆, were isolated from Gordona lentifragmenta and from G. bronchialis, respectively. On the other hand, G. rubropertincta showed only a monoacylglycerol fraction which released non-hydroxylated fatty acids; they were identified as C_16:0-, C_16:1,- C_18:1- and branched C_19:0-fatty acids. This last component was identified as 10-methyl octadecanoic acid (tuberculostearic acid).     

Gibberellins and the Legume-Rhizobium Symbiosis : I. Endogenous Gibberellins of Lima Bean (Phaseolus lunatus L.) Stems and Nodules

The content of gibberellin-like substances in nodules formed by Bradyrhizobium species strain 127E14 on roots of lima bean (Phaseolus lunatus L.) has been previously found to be relatively high. The objectives of the present study were to purify and identify the endogenous gibberellins from the stems and nodules of lima bean. By sequential silica gel partition column chromatography, C₁₈ reverse-phase high performance liquid chromatography, and combined gas chromatography-mass spectrometry, the gibberellins A₁, A₃, A₁₉, A₂₀, A₂₉, and A₄₄ were identified from root nodules. Gibberellins A₁, A₃, A₁₉, A₂₀, and A₄₄ were also identified from lima bean stem tissue. These data provide the first mass spectral-based evidence that gibberellins are present in leguminous root nodules. The presence of the gibberellins identified indicates that the early 13-hydroxylation gibberellin biosynthetic pathway predominates in stem and nodule tissue. However, it is not known if the gibberellins within the nodules are produced in situ, or if they are imported from some remote host plant tissue.     

Identification of gibberellins A17, A25, A45, abscisic acid,phaseic acid,and dihydrophaseic acid in seeds of Pyrus communis

Gibberellin A₁₇, abscisic acid, and 4′-dihydrophaseic acid were identified by GC-MS of derivatized extracts from both immature and mature seeds of pear. Immature seeds also contained phaseic acid, gibberellins A₂₅ and A₄₅, and two presumed mono-hydroxylated derivatives of GA₄₅, one of which was tentatively identified as 3β-hydroxy-GA₄₅. Several presumed metabolites of abscisic acid were detected in both mature and immature seeds.     

Identification of Endogenous Gibberellins in Dormant Bulbils of Chinese Yam, Dioscorea opposita

It is known that dormancy of the genus Dioscorea is induced by application of gibberellin (GA) A₃. To understand the role of GAs in dormancy induction, endogenous GAs have been identified by Kovats retention indices and full mass spectra from capillary gas chromatography-mass spectrometry analysis of purified extract from dormant bulbils of Dioscorea opposita Thunb. These include GA₄, GA₉, GA₁₂, GA₁₉, GA₂₀, GA₂₄, GA₃₆, and GA₅₃; their presence suggests the occurrence of two biosynthetic pathways in D. opposita bulbils, the early 13-hydroxylation pathway and the non-13-hydroxylation pathway.     

Identification of Endogenous Gibberellins from Salix pentandra

Gibberellins A₁, A₁₉, A₂₀, and A₂₉ have been identified by sequential high-performance liquid chromatography retention time (Rt) and combined gas chromatography-mass spectrometry (Rt and characteristic mass spectra) from elongating shoots of Salix pentandra L. Gibberellins A₁ and A₁₉ were also detected in purified extracts from male and female flowers (catkins) of S. pentandra.     

Gibberellins inCitrus sinensis: A comparison between seeded and seedless varieties

The gibberellin (GA) content of the reproductive organs ofCitrus sinensis (L.) Osb., cv. Bianca Comuna and the seedless variety, Salustiana, were examined by combined gas chromatography-mass spectrometry (GC/MS) at different stages of development. Gibberellins A₁, A₂₀, and A₂₉ were identified in the reproductive buds of both cultivars 21 days prior to anthesis and in fruits 35 days after anthesis by comparison of their mass spectra and Kovats retention indices with those of standards. In addition, three uncharacterized isomers of GA₁ were detected, one in buds and two in fruits. The presence of GA₄ in both tissues, and of GA₈ in the reproductive buds, was indicated by the occurrence of characteristic ions at the expected retention times, although their spectra were too weak for full identification. Vegetative shoots of cv. Salustiana contained gibberellins A₁, A₁₉, A₂₀, and A₂₉, and the unidentified isomer of GA₁ present in reproductive buds. The presence of trace amounts of gibberellins A₈ and A₁₇ was also indicated. Although the two varieties did not differ qualitatively in the GAs present during flower and fruit development, the seedless variety contained slightly greater amounts. The concentrations of gibberellins A₁, A₄, and A₂₀ were determined by gas chromatography-selected ion monitoring (GC/SIM) throughout ovary development and early fruit growth. In both varieties, the maximum GA₁ concentration occurred at anthesis. Highest concentrations of gibberellins A₂₀ and A₄ were found in fruit 35 days after anthesis, although the GA₁ concentration at this stage remained low.     

Native gibberellins and the metabolism of [14C]gibberellin A53 and of [17-13C, 17-3H2]gibberellin A20 in tassels of Zea mays

The native hormones from tassels of maize (Zea mays) were re-investigated. The previous identification by GC/SIM of GA₁, GA₈ and GA₂₉ in normal tassels was confirmed by full GC/MS scans at the correct Kovats retention indices. In tassels of dwarf-1 mutants, GA₄₄,^?GA₁₉, GA₁₇, GA₂₀ and the 16,17-dihydro, 7β,16α,17-trihydroxy derivative of ent-kaurenoic acid were identified by GC/MS. Gibberellin A₁ was not found in the mutant tassels. [¹⁴C]Gibberellin A₅₃ was fed to tassels of the dwarf-5 mutant. In the ethyl acetate-soluble acidic fraction from the feeds, [¹⁴C]GA₄₄ was identified by GC/MS; [¹⁴C]GA₁₉ and [¹⁴C]GA₂₉ were identified by GC/SIM. The GA₂₉ is probably a metabolite of the feeds because the dwarf-5 mutant is known to control the step copalyl pyrophosphate to ent-kaurene in the maize GA-biosynthetic pathway and because GA₂₉ was not identified in a control experiment. The n-butanol fractions obtained from the feeds were shown, by GC/MS, to contain [¹⁴C]GA₅₃ after hydrolysis, suggesting that conjugated [¹⁴C]GA₅₃ is a major metabolite from GA₅₃ feeds. [17-¹³C, 17-³H₂]Gibberellin A₂₀ was fed to normal, dwarf-1 and dwarf-5 tassels. In each case, analysis of the purified ethyl acetate-soluble acidic extracts by GC/MS led to the identification of [¹³C]GA₂₉ and unmetabolized [¹³C]GA₂₀ in which no ¹³C-isotope dilution was observed.     

Synthesis and antiviral activity of novel glycyrrhizic acid conjugates with D-amino acid esters

Glycyrrhizic acid (GA) conjugates with methyl and ethyl esters of D-amino acids (D-Trp, D-Phe, D-Tyr, D-Val, D-Leu) have been synthesized by the activated esters method using mixtures of N-hydroxybenzotriazole or N-hydroxysuccinimide with N,N′-dicyclohexylcarbodiimide. GA conjugate with D-Trp ethyl ester exhibited antiviral activity against influenza viruses A/H3N2, A/H1N1/pdm09, A/H5N1, B (SI > 10–29), and HRSV (SI > 25). GA conjugate with D-Trp methyl ester inhibited influenza virus A/H1N1/pdm09 (SI > 30).     

Diterpene acids from Helianthus species and their microbiological conversion by Gibberella fujikuroi, mutant B1-41a

The diterpene acid content in 10 species of Helianthus has been investigated. Ent-12,16-cyclokauranoic acid, isolated from H. annuus, is converted into a series of 12,16-cyclogibberellins by cultures of Gibberella fujikuroi, mutant B1-41a, and 12,16-cyclogibberellins A₉, and A₁₂ have been isolated. Ent-12β-acetoxykaurenoic acid and ent-13(S)-angeloxyatisenoic acid have been isolated from H. decapetalus; the metabolism of ent-13(S)-hydroxyatisenoic acid and atisenoic acid by B1-41a is also described.     

Metabolism of Gibberellin A(12) and A(12)-Aldehyde in Developing Seeds of Pisum sativum L

Metabolism of [¹⁴C]gibberellin (GA) A₁₂ (GA₁₂) and [¹⁴C]gibberellin A₁₂-aldehyde (GA₁₂-aldehyde) was examined in cotyledons and seed coats from developing seeds of pea (Pisum sativum L.). Both were metabolized to only 13-hydroxylated GAs in cotyledons but to 13-hydroxylated and non-13-hydroxylated GAs in seed coats. The metabolism of [¹⁴C]GA₁₂ was slower in seed coats than in cotyledons. [¹⁴C]GA₁₂-aldehyde was also metabolized to conjugates in seed coats. Seed coat [¹⁴C]-metabolites produced from [¹⁴C]GA₁₂-aldehyde were isolated by high-performance liquid chromatography (HPLC). Conjugates were base hydrolyzed and the free GAs reisolated by HPLC and identified by gas chromatography-mass spectrometry. [¹⁴C]GA₅₃-aldehyde, [¹⁴C]GA₁₂-aldehyde conjugate, and [¹⁴C]GA₅₃-aldehyde conjugate were major metabolites produced from [¹⁴C]GA₁₂-aldehyde by seed coats aged 20-22 days or older. The dilution of ¹⁴C in these compounds by ¹²C, as compared to the supplied [¹⁴C]GA₁₂-aldehyde, indicated that they are endogenous. Feeding [¹⁴C]GA₅₃-aldehyde led to the production of [¹⁴C]GA₅₃-aldehyde conjugate in seed coats and shoots and also to 13-hydroxylated GAs in shoots. Labeled GAs, recovered from plant tissue incubated with either [¹⁴C]GA₁₂, [¹⁴C]GA₁₂-aldehyde, or [³H]GA₉, were used as appropriate markers for the recovery of endogenous GAs from seed coats or cotyledons. These GAs were purified by HPLC and identified and quantified by gas chromatography-mass spectrometry. GA₁₅, GA₂₄, GA₉, GA₅₁, GA₅₁-catabolite, GA₂₀, GA₂₉, and GA₂₉-catabolite were detected in seed coats, whereas GA₉, GA₅₃, GA₄₄, GA₁₉, GA₂₀, and GA₂₉ were found in cotyledons. The highest GA levels were for GA₂₀ and GA₂₉ in cotyledons (783 and 912 nanograms per gram fresh weight, respectively) and for GA₂₉ and GA₂₉-catabolite in seed coats (1940 and > 1940 nanograms per gram fresh weight, respectively).     

Characterization of gibberellins from light-grown Phaseolus coccineus seedlings by combined GC-MS

Following extensive purification of an extract from 5000 light-grown Phaseolus coccineus seedlings by procedures including countercurrent distribution, Sephadex G10, polyvinylpyrrolidone, charcoal-celite and silicic acid partition column chromatography, TLC preparative GLC, gibberellins A₁, A₄, A₅ and A₂₀ were characterized by combined GC-MS. In addition, an unknown compound isomeric with gibberellin A₁₇ but lacking an hydroxyl group, was also detected.