Identification of gibberellin A4 in Pisum sativum L. and the effects of applied gibberellins A9, A4, A5 and A3 on the le mutant

Gibberellin A₄ (GA₄) was identified for the first time in the garden pea (Pisum sativum) L.), by gas chromatography-mass spectrometry. However, in wild-type shoots the level of GA₄ was only about 6% of the level of GA₁, and it is therefore unlikely that GA₄ plays a major role per se in the control of pea stem elongation. In shoots of the le mutant, GA₄ was not detected, while the level of GA₉ was approximately twice that found in the wild-type. The le mutation also markedly reduced the elongation response to applied GA₉. It appears, therefore, that in Pisum the le mutation blocks the 3-hydroxylation of GA₉ to GA₄, in addition to the 3-hydroxylation of GA₂₀ to GA₁. In contrast, the le mutation did not reduce the response to applied GA₅, suggesting the step GA₅ to GA₃ is not catalysed by the enzyme controlled by the Le gene. The step GA₅ to GA₃ was confirmed in peas by metabolite analysis after treatment with deuterated GA₅.     

Responses of florence fennel (Foeniculum vulgare azoricum) seeds to light,temperature and gibberellin A4/7

The percentage and the rate of germination of seeds of three varieties of Florence fennel was higher in the dark than in the light, the high temperature cut-off points being between 27.2 and 29.4°C. The optimum temperature for germination was between 20 and 25°C. Seeds of all three varieties responded to incubation in solutions containing gibberellin A_4/7 mixture (GA_4/7; Regulex), giving higher germination in the light at temperatures from 20 to 30°C. Seeds steeped for 4 h at 25°C or for 24 h at 5°C in GA_4/7 solutions gave a higher percentage and increased rate of emergence as compared with untreated dry seeds, when sown in compost at 25°C; steeping in water alone was also beneficial. In general, drying the treated seeds before sowing reduced the rate but sometimes increased the percentage of germination as compared with seeds sown when still moist. Seeds harvested from secondary umbels of var. Zefa fino germinated better both in the light and dark than those taken from primary umbels.Abbreviations GA_4/7 a commercial mixture of the gibberellins A₄ and A₇ (trade name Regulex)     

Convergent pathways of gibberellin A1 biosynthesis in Brassica

[²H, ³H]Gibberellin A₄ (GA₄) or [²H, ³H] GA₉ were applied to the shoot tips of seedlings of elongated internode (ein), a tall mutant of rapid cycling Brassica rapa. Following [²H]GA₉ application, [²H]GA₅₁, [²H]GA₂₀ and [²H]GA₄ were identified as products by GC-MS, while [²H]GA₃₄ and [²H]GA₁ were formed from [²H]GA₄. Other isotopically labelled products, including abundant putative conjugates, were also produced, but were not identified. Thus, in B. rapa, GA₁ biosynthesis involves the convergence of at least two metabolic pathways; it can be formed via GA₄ or GA₂₀, the latter of which can originate from GA₉ or from GA₁₉.     

Enzyme-linked immunosorbent assay for the activator protein specific for the enzymic hydrolysis of GM1 in urine

We have established a sensitive and specific enzyme-linked immunosorbent assay (ELISA) for the detection of the activator protein which stimulates the enzymic hydrolysis of G_M1 (G_M1-activator) in human urine. The level of G_M1-activator in 19 normal, adult urine samples was estimated to be 370.7±33.2 ng/ml. The amounts of G_M1-activator excreted in 24 h were estimated to be between 0.28 and 1.1 mg. The coefficient of variation for this method is 4.3% for the intra-assay and 14.4% for the inter-assay. Urine samples, without purification, can be used directly for the ELISA.     

Promotion of flowering in apple trees with gibberellin A4 and C-3 epi-gibberellin A4

The proportion of spurs flowering on apple trees (Malus domestica Borkh. cv Golden Delicious) displaying a high degree of alternate-year flowering was increased in the off year by gibberellin A₄ (GA₄) and C-3 epi-GA₄ applied in the previous year. When applied 4.5 weeks after anthesis amounts of GA₄ ranging from 3 to 300 g per spur and 25 or 50 g of C-3 epi-GA₄ per spur were effective. Treatments with GA₄ made seven weeks after anthesis were less effective. A combination of 30 g GA₄ and 30 g zeatin (6-(4-hydroxy-3-methylbut-trans-2-enylamino)purine) promoted flowering at both treatment times, and tended to be more effective than GA₄ alone.Abbreviation GA gibberellin or gibberellin-like substance Contribution No. 618     

Development and evaluation of an enzyme-linked immunosorbent assay to detect Pieris rapae remains in guts of arthropod predators

An enzyme-linked immunosorbent assay (ELISA) was developed to detect remains of Pieris rapae L. (Lepidoptera: Pieridae) immature stages in the guts of field collected arthropod predators. The assay can be used to help ascertain the relative importance of arthropod predator species in suppressing P. rapae in cabbage, Brassica oleracea var. capitata L. The ELISA is sensitive to all immature stages of P. rapae, although first and fifth instars can be detected more readily than eggs or pupae and third instars showed intermediate detectability. Assays on whole body homogenates of predators readily detected predation on P. rapae first instars by all seven of the predator species tested, although response generally declined with increasing predator size. Together the results show that the P. rapae ELISA possesses a sufficiently high level of sensitivity and specificity to be a useful tool in helping to elucidate the roles of arthropod predator species in reducing populations of P. rapae in cabbage.     

Ontogenetic variation in levels of gibberellin A1 in Pisum

The levels of the biologically active gibberellin (GA), GA₁, and of its precursor, GA₂₀, were monitored at several stages during ontogeny in the apical portions of isogenic tall (Le) and dwarf (le) peas (Pisum sativum L.) using deuterated internal standards and gas chromatography-selected ion monitoring. The levels of both GAs were relatively low on emergence and on impending apical arrest. At these early and late stages of development the internodes were substantially shorter than at intermediate stages, but were capable of large responses to applied GA₃. Tall plants generally contained 10–18 times more GA₁ and possessed internodes 2–3 times longer than dwarf plants. Further, dwarf plants contained 3–5 times more GA₂₀ than tall plants. No conclusive evidence for the presence of GA₃ or GA₅ could be obtained, even with the aid of [²H₂]GA₃ and [²H₂]GA₅ internal standards. If GA₃ and GA₅ were present in tall plants, their levels were less than 0.5% and 1.4% of the level of GA₁, respectively. Comparison of the effects of gene le on GA₁ levels and internode length with the effects of ontogeny on these variables shows that the ontogenetic variation in GA₁ content was sufficient to account for much of the observed variation in internode length within the wild-type. However, evidence was also obtained for substantial differences in the potential length of different internodes even when saturating levels of exogenous GA₃ were present.Abreviations GA_n gibberellin A_n We thank Noel Davies, Omar Hasan, Leigh Johnson, Katherine McPherson and Naomi Lawrence for technical help, Professor L. Mander (Australian National University, Canberra) for deuterated GA standards and the Australian Research Council for financial assistance.     

Synthesis of N-alkyl glycine amides as potent inhibitors of leukotriene A4 hydrolase

The synthesis and biological evaluation of a series of N-alkyl glycine amide analogs as LTA₄-h inhibitors and the importance of the introduction of a benzoic acid group to the potency and pharmacokinetic parameters of our analogs are described. The lead compound in the series, 4q, has excellent potency and oral bioavailability.     

Persistence of 14C-gibberellin A3 on the surface of Citrus fruit peel and on inert glass surfaces

The persistence of gibberellin A₃ on plant surfaces was examined using fruit of Marsh seedless grapefruit (Citrus paradisi Macf.) and an inert glass model system. ¹⁴C-gibberellin A₃ was applied to surfaces in aqueous treatment solutions or in waxing solutions. Dried-out treatment residues were removed by washing and analyzed for total and GA₃-like radioactivity. Gibberellin A₃ persisted without significant loss for at least 7 d in aqueous treatment solutions (pH 4.0 or 6.2) but was less persistent in the pH 10.4 waxing solution (t1/2=7 d).Loss of total peel surface radioactivity was fast during the first 3 days, slowing down afterwards. After 14 days 73% of the initial radioactivity could still be recovered from fruit peel surface and 70% of the recovered radioactivity was still in the form of gibberellin A₃. Gibberellin A₃ was somewhat more persistent in residues from pH 4 than pH 7 treatment solutions. Light had a slight enhancing effect on gibberellin A₃ decomposition on fruit peel under growth chamber conditions. After 12 d at 100% relative humidity, 88% of the radioactivity on glass surfaces was still in the form of gibberellin A₃, as against 45% at a relative humidity of 50%. Simulated field conditions, combining daily fluctuations in light, temperature and relative humidity, markedly enhanced gibberellin A₃ decomposition on glass surfaces (t1/2=2 d). Gibberellin A₃ was very persistent (90% after 9 d) in the waxing residues on fruit peel surface.Abbreviations GA₃ gibberellin A₃ - RH relative humidity     

A colorimetric sandwich-type assay for sensitive thrombin detection based on enzyme-linked aptamer assay

A colorimetric sandwich-type assay based on enzyme-linked aptamer assay has been developed for the fast and sensitive detection of as low as 25 fM of thrombin with high linearity. Aptamer-immobilized glass was used to capture the target analyte, whereas a second aptamer, functionalized with horseradish peroxidase (HRP), was employed for the conventional 3,5,3′,5′-tetramethylbenzidine (TMB)-based colorimetric detection. Without the troublesome antibody requirement of the conventional enzyme-linked immunosorbent assay (ELISA), as low as 25 fM of thrombin could be rapidly and reproducibly detected. This assay has superior, or at least equal, recovery and accuracy to that of conventional antibody-based ELISA.     

Discovery of novel and potent aryl diamines as leukotriene A4 hydrolase inhibitors

The synthesis and biological evaluation of a series of aryl diamines as inhibitors of LTA₄-h inhibitors are described. The optimization which led to the identification of the optimal para-substitution on the diphenyl ether moiety and diamine spacer is discussed. The resulting compounds such as 3l have excellent enzyme and cellular potency as well as desirable pharmacokinetic properties.     

A new non-equilibrium enzyme linked immunosorbent assay for a glycogen-derived urinary tetrasaccharide

The tetrasaccharide, Glc1-6Glc1-4Glc1-4Glc, denoted (Glc)₄, is a limit dextrin formed by amylolytic degradation of glycogen. In order to evaluate the possible clinical importance of (Glc)₄ excretion as an indicator of certain physiological and pathological conditions, we have developed a new rapid and inexpensive immunoassay using a monoclonal antibody raised against (Glc)₄ glycosidically-linked to a carrier protein. As the antibody is highly specific, it can be used to measure native (Glc)₄ directly, without the chemical reduction step required in previous methods. A new type of non-equilibrium ELISA inhibition test was developed based on the capacity of free (Glc)₄ to decrease initial rates of antibody binding to (Glc)₄-coated microtiter wells. The method is highly reproducible and is as sensitive and accurate as the gas chromatography method or radioimmunoassay used previously.Abbreviations (Glc)₄ Glc1-6Glc1-4Glc1-4Glc - KLH keyhole limpet hemacyanin - BSA bovine serum albumin - PEG polyethylene glycol     

Development of a one-step enzyme immunoassay for the quantitation of gibberellin A3 in barley seeds

The development of a sensitive and specific enzyme immunoassay for GA₃ is reported. This method was based on the use of peroxidase labelled GA₃ and immobilized antibodies. In order to obtain a rapid immunoassay, several steps of purification were analyzed to show their necessity. Barley seed extracts were assayed at different steps of purification to exhibit the effect of extract components on the assay. It was demonstrated that HPLC had to be performed when a selective quantitation of GA₃ was required. This assay allowed GA₃ to be measured with reproducibility as its unmethylated form and the quantitation of GA₃ in barley seeds with this enzyme immunoassay was correlated to a GC-MS method.Abbreviations GA₃ gibberellin A₃ - EIA enzyme immunoassay - DMF dimethylformamide - TEA tri(n)ethylamine - BSA bovine serum albumin - OVA ovalbumine - ECF ethylchloroformate - PB phosphate buffer     

A non-invasive assay of the plastoquinone pool redox state based on the OJIP-transient

The plastoquinone (PQ) pool of the photosynthetic electron transport chain becomes reduced under anaerobic conditions. Here, anaerobiosis was used as a tool to manipulate the PQ-pool redox state in darkness and to study the effects of the PQ-redox state on the Chl-a fluorescence (OJIP) kinetics in pea leaves (Pisum sativum L.). It is shown that the F_J (fluorescence intensity at 3 ms) is linearly related to the area above the OJ-phase (first 3 ms) representing the reduction of the acceptor side of photosystem II (PSII) and F_J is also linearly related to the area above the JI-phase (3–30 ms) that parallels the reduction of the PQ-pool. This means that F_J depends on the availability of oxidized PQ-molecules bound to the Q_B-site. The linear relationships between F_J and the two areas indicate that F_J is not sensitive to energy transfer between PSII-antennae (connectivity). It is further shown that a ∼94% reduced PQ-pool is in equilibrium with a ∼19% reduction of Q_A (primary quinone acceptor of PSII). The non-linear relationship between the initial fluorescence value (F_{20 μs}) and the area above the OJ-phase supports the idea that F_{20 μs} is sensitive to connectivity. This is reinforced by the observation that this non-linearity can be overcome by transforming the F_{20 μs}-values into [Q_A ⁻]-values. Based on the F_J-value of the OJIP-transient, a simple method for the quantification of the redox state of the PQ-pool is proposed. Szilvia Z. Tóth and Gert Schansker contributed equally to this study.     

Stimulatory effect of ethephon,ACC, gibberellin A3 and A4+7 on germination of methyl jasmonate inhibited Amaranthus caudatus L. seeds

Methyl jasmonate (JA-Me) inhibited or retarded germination of Amaranthus caudatus seeds in darkness at 24°C, Ethephon, ACC and gibberellins (GA₃ or GA₄₊₇) partially or completely reversed this inhibition depending on the concentration of JA-Me applied. Both ethephon and the gibberellins were more effective than ACC. Both GA₃ and GA₄₊₇ enhanced the stimulatory effect of ethephon or ACC on germination of seeds inhibited by JA-Me.Abbreviations ACC 1-aminocyclopropane-1-carboxylic acid - JA jasmonic acid - JA-Me methyl jasmonate     

Identification and characterization of a novel secreted antigen 1 of Babesia microti and evaluation of its potential use in enzyme-linked immunosorbent assay and immunochromatographic test

Here, we identified a novel secreted antigen designated as Babesia microti secreted antigen 1 (BmSA1) by immunoscreening a B. microti cDNA expression library using the sera from hamsters immunized with plasma, putatively containing secreted antigens, from B. microti-infected hamsters. Antibodies raised in mice immunized with recombinant BmSA1 (rBmSA1) recognized a native 33-kDa parasite protein. An enzyme-linked immunosorbent assay (ELISA) of rBmSA1 detected specific antibodies as early as 6 and 4 days post-infection in sera from a hamster experimentally infected with B. microti Gray strain (US type) and a mouse experimentally infected with B. microti Munich strain (rodent isolate), respectively. Moreover, a rapid immunochromatographic test (ICT) using rBmSA1 detected specific antibodies in a hamster experimentally infected with B. microti from day 6 to at least day 270 post-infection, which was quite consistent with the results of the ELISA. In addition, analysis of the sera involved in the first case of human babesiosis in Japan (Kobe type) showed that specific antibodies were detectable in the patient and the positive donor by ELISA using rBmSA1, and the ICT result was identical to the ELISA data. Taken together, these results indicated that BmSA1 could be a promising and universal target for developing both ELISA and ICT for the serodiagnosis of human babesiosis and for an epidemiological survey of its rodent reservoir.     

Characterization of monoclonal antibodies to protoplast membranes of Nicotiana tabacum identified by an enzyme-linked immunosorbent assay

Murine monoclonal antibodies to protoplast membrne antigens were generated using mouse myelomas and spleen cells from mice immunized with Nicotiana tabacum L. leaf protoplasts. For selecting antibody-secreting clones, a sensitive and rapid enzyme-linked immunosorbent assay (ELISA) for monoclonal antibody binding to immobilized cellular membrane preparations or immobilized protoplasts was developed. With intact protoplasts as immobilized antigen, the ELISA is selective for antibodies that bind to plasma-membrane epitopes present on the external surface of protoplasts. Using the membrane ELISA, a total of 24 hybridoma lines were identified that secreted antibodies to plant membrane epitopes. The protoplast ELISA and subsequent immunofluorescence studies identified four hybridoma lines as secreting antibodies which bound to the external surface of protoplasts and cells. The corresponding antigens were not species- or tissue-specific, were periodatesensitive, and were located in membranes which equilibrated broadly throughout a linear sucrose gradient. When protein blots of electrophoretically separated membrane proteins were probed with these antibodies, a band of M_r 14 kilodaltons (kDa) and a smear of bands of M_r 45–120 kDa were labeled. An additional set of three antibodies appeared by immunofluorescence to bind to the plasma membrane of broken but not intact protoplasts and labeled membranes equilibrating at a density of approx. 1.12 kg·l^-1 in a linear sucrose density gradient. These classes of monoclonal antibodies enlarge the library of monoclonal antibodies (Norman et al. 1986, Planta 167, 452–459) available for the study of plant plasma-membrane structure and function.Abbreviations ELISA Enzyme-linked immunosorbent assay - Ig immunoglobulin - kDa kilodalton - M_r relative molecular mass - SDS-PAGE sodium dodecyl sulfate polyacrylamide gel electrophoresis     

Serum leucine-rich alpha-2-glycoprotein-1 binds cytochrome c and inhibits antibody detection of this apoptotic marker in enzyme-linked immunosorbent assay

Cytochrome c (Cyt c) has been implicated as a serum marker for aberrant apoptosis and, thus, has considerable clinical potential. Using a sandwich enzyme-linked immunosorbent assay (ELISA) we found that the sensitivity of Cyt c detection is reduced in the presence of serum. The inhibitory factor responsible was purified from both fetal bovine serum and human serum employing standard chromatography procedures followed by affinity chromatography on Affi-Gel 10-bound Cyt c. In SDS-PAGE, bands at 44 kD and 50 kD were observed for the bovine and human proteins, respectively. Mass spectrometry analysis identified the serum inhibitory factor as leucine-rich alpha-2-glycoprotein-1 (LRα2GP1). This identification may lead to a modified ELISA to quantify total Cyt c in patients’ sera. LRα2GP1 is the first extracellular ligand for Cyt c that has been identified. A physiological function associated with binding is suggested. Supported by: NIH NINDS 5R21-NS45589     

Endogenous ethylene and reversing methyl jasmonate inhibition of Amaranthus caudatus seed germination by benzyladenine or gibberellin

BA at 10^–5 M, GA₃ at 3×10^–4 M or GA₄₊₇ at 3×10^–5 M partially or largely reversed the inhibition of Amaranthus caudatus seed germination due to JA-Me. BA or GA₃ did not affect ethylene production and ACC oxidase activity in vivo in the presence of JA-Me before radicle protrusion. However, both increased ethylene production after 72 h of incubation, when the reversal of the JA-Me inhibition of seed germination was observed. AVG at 3×10^–4 M decreased ethylene production when it was applied simultaneously with BA and JA-Me or GA₃ and JA-Me, but it had no effect on seed germination. NBD almost completely reversed the stimulatory effect of BA, GA₃ or GA₄₊₇ on the germination of seeds in the presence of JA-Me. Exogenous ethylene reversed the inhibitory effect of NBD. The results indicate that action of endogenous ethylene is involved in the response of JA-Me inhibited seeds to BA or GA_s.     

Involvement of endogenous gibberellins in the regulation of increased tomato shoot growth in solarized soil

Environmental factors often affect plant growth bymodifying the levels of endogenous gibberellins (GAs).In this study, the involvement of GAs in theregulation of enhanced shoot growth in tomato (Lycopersicon esculentum Mill.) plants grown in soiltreated by solarization (a soil disinfestation method)was investigated. Seedlings at the cotyledonary stagewere transplanted into either solarized or untreatedcontrol soil. Plants in both soils grew free of anydisease symptoms. As soon as four days after planting,seedlings in solarized soil had a higher dry weightcompared to the control. Throughout most of theexperimental period of 18 days, leaf weight ratio washigher in the solarized vs. the control soil. Treatingshoot tips of control plants with 0.1 mg^.L^-1GA₃ resulted in enhanced leaf and stem growth,thus reaching values similar to those of plants grownin solarized soil. The opposite effect was obtained bytreating plants grown in solarized soil with1 mg^.L^-1 uniconazole, a GA biosynthesisinhibitor. Quantitative GC-MS analyses revealed thatGA₁ content in one and two-weeks old transplantsgrown in various solarized soils was up to 1.8 fold,and that GA₃ content in two-weeks old plants wasup to five fold the values in the control. Theseincreases were linearly correlated with the increasein leaf dry weight. It was concluded that theincreased quantities of GA₁, and eventuallyGA₃, play a role in the increased growth oftomato shoots in solarized soil as early as seven daysafter transplanting.deceased