Preparation of Radioactive Gibberellins A20, A5 and A8

Many yeasts were isolated from natural sources in the tropics and subtropics by enrichment culture technique, using medium which contained a surfactant. The medium was acidified with citric acid. A strain S–10 belonging to the genus Candida was found to produce itaconic acid. Under suitable conditions in shake culture, a mutant derived from this strain produced the acid at about 35 % yield on the basis of glucose supplied.     

Radioimmunoassay of plasma 20 alpha-dihydroprogesterone

A radioimmunological method is described and evaluated for the determination of 20α-dihydroprogesterone (20α-hydroxypregn-4-en-3-one) in peripheral venous plasma (0.1 – 2.O ml) of men and women. The antiserum is highly specific. The concentration (mean ± S.D., ng/ml plasma) in a group of healthy men was 0.19 ± 0.08; in women in follicular phase, 0.28 ± 0.11; in luteal phase, 2. 19 ± 1.19; and during pregnancy (24th week to term) in the range 8.44– 40.94.     

Radioimmunoassay of 3 alpha-hydroxy-5 alpha-pregnan-20-one in rat and human plasma

A radioimmunoassay for measuring 3 alpha-hydroxy-5 alpha-pregnan-20-one in plasma has been developed. Polyclonal antibodies were raised in rabbits against 3 alpha-hydroxy-20-oxo-5 alpha-pregnan-11 alpha-yl carboxymethyl ether coupled to bovine serum albumin. 3 alpha-Hydroxy-5 alpha-pregnan-20-one was purified from either extracts of plasma by high-performance liquid chromatography. These antibodies were then used for the radioimmunoassay of this centrally active progesterone metabolite in rat and human plasma. 3 alpha-Hydroxy-5 alpha-pregnan-20-one was detected in plasma from female rats on the day of estrus (2.0 to 9.3 ng/ml) and in the plasma of women during the luteal phase of the menstrual cycle at levels ranging from 0.25 to 2.5 ng/ml. The latter was highly correlated with plasma progesterone levels.     

Isolation and Characterization of Gibberellins in Calonyction aculeatum and Structures of Gibberellins A30, A31, A33 and A34

Four novel gibberellins GA₃₀, GA₃₁, GA₃₃, GA₃₄, five known gibberellins GA₈, GA₁₇ (free acid and its monomethyl ester), GA₁₉, GA₂₇, GA₂₉ and the gibberellin-like substances were isolated from immature seeds of evening-glory (Calonyction aculeatum). The structures of GA₃₀, GA₃₁, GA₃₃ and GA₃₄ were elucidated as IX, XVIII, XXII and XXXIV, respectively. The three gibberellin-like substances were partially characterized.     

A simple synthesis of 3beta-acetoxy-20-oxomethylpregn-5-ene and 3beta-acetoxy-20-hydroxymethylpregn-5-ene

3beta-Acetoxy-20-oxomethylpregn-5-ene and 3beta-acetoxy-20-hydroxymethylpregn-5-ene were synthesized from (22R,23R)-sitost-5-ene-3beta,22,23-triol in 66% overall yields.     

Gibberellins of sugarcane

In our hands a phosphate buffered celite column has given an adequate separation of GA₁ and GA₃. These 2 gibberellins are normally very difficult to separate. Young sugarcane growing under moisture stress contains at least 2 gibberellin-like substances. One is suspected to be GA₅. The other is unknown but has high activity in the barley endosperm assay and is neither GA₁ nor GA₃. Four-month-old cane contains 2 major growth promoters. From their chromatographic, fluorimetric and biological properties these are thought to be GA₁ and GA₃. Rapidly growing 6-month-old cane has a surprisingly low level of gibberellin-like substances.     

Radioimmunoassay for orexin A

A radioimmunoassay for orexin A has been developed. Anti-orexin A antiserum was raised in New Zealand white rabbits immunized with a conjugate of synthetic orexin A with bovine serum albumin. This antibody did not crossreact with orexin B, hypothalamic hormones, pituitary hormones, neuropeptides or gut hormones. Radioiodination of orexin A was performed with the chloramin T method, followed by purification of radioiodinated material on Sephadex G-25 column. Orexin A was extracted from tissues using acid-acetone. The assay was performed with a double antibody system. The dilution curve of acid-acetone-extracts of rat hypothalamus in the radioimmunoassay system was parallel to the standard curve. The recovery of tissue orexin A was about 80%,and the intra-assay and inter-assay variations were 5.2% and 7.8%, respectively. Orexin A was found in the hypothalamus, cerebrum and testis. These data suggest that this assay system is suitable for the measurement of tissue orexin A and that orexin A is found in the central nervous system and testis.     

Radioimmunoassay of the A prostaglandins

Antibodies to the (PGA) prostaglandin A were produced in rabbits immunized with a conjugate of PGE2 covalently linked to (BSA) bovine serum albumin by reaction with carbodiimide reagent. A radioimmunoassay was developed using dextran-coated charcoal to separate the free from antibody bound PGA1-3H. The sensitivity of the method was found to be 100 picograms/ml of plasma. Ethyl acetate was used for extraction of plasma and the various classes of PGs were separated by silicic acid column chromatography. Recovery of PGA1-3H throughout the entire procedure was 65-75%. The antibody showed progressively decreasing affinity to PGA2, PGA1, PGE2, PGE1, PGB2, and PGF2alpha, respectively. The mean plasma PGA level in adult males (N=13) was found to be 1.39 + or - 0.55 ng/ml, and 1.62 + or - 0.52 ng/ml in adult females (N=7). Corresponding plasma and serum samples were found to give essentially similar results. Plasma PGA levels in adult males treated with indomethacin for rheumatoid arthritis were 0.18 + or - 0.15 ng/ml (P 0.001 in comparison with the normal adult males). This method is sufficiently sensitive, precise, and rapid to allow the routine estimation of the PGAs in biological samples.     

Gibberellins and Gravitropism in Maize Shoots : Endogenous Gibberellin-Like Substances and Movement and Metabolism of [3H]Gibberellin A20

[³H]Gibberellin A₂₀ (GA₂₀) of high specific radioactivity (49.9 gigabecquerel per millimole) was applied equilaterally in a ring of microdrops to the internodal pulvinus of shoots of 3-week-old gravistimulated and vertical normal maize (Zea mays L.), and to a pleiogravitropic (prostrate) maize mutant, lazy (la). All plants converted the [³H]GA₂₀ to [³H]GA₁⁻ and [³H]GA₂₉-like metabolites as well as to several metabolites with the partitioning and chromatographic behavior of glucosyl conjugates of [³H]GA₁, [³H]GA₂₉, and [³H]GA₈. The tentative identification of these putative [³H]GA glucosyl conjugates was further supported by the release of the free [³H]GA moiety after cleavage with cellulase. Within 12 hours of the [³H]GA₂₀ feed, there was a significantly higher proportion of total radioactivity in lower than in upper halves of internode and leaf sheath pulvini in gravistimulated normal maize. Further, there was a significantly higher proportion of putative free GA metabolites of [³H]GA₂₀, especially [³H]GA₁, in the lower halves of normal maize relative to upper halves. The differential localization of the metabolites between upper and lower halves was not apparent in the pleiogravitropic mutant, la. Endogenous GA-like substances were also examined in gravistimulated maize shoots. Forty-eight hours after gravistimulation of 3-week-old maize seedlings, endogenous free GA-like substances in upper and lower leaf sheath and internode pulvini halves were extracted, chromatographed, and bioassayed using the `Tanginbozu' dwarf rice microdrop assay. Lower halves contained consistently higher total levels of GA-like activity. The qualitative elution profile of GA-like substances differed consistently, upper halves containing principally a GA₂₀-like substance and lower halves containing mainly GA₁-like and GA₁₉-like substances. Gibberellins A₁ (10 nanograms per gram) and A₂₀ (5 nanograms per gram) were identified from these lower leaf sheath pulvini by capillary gas chromatography-selected ion monitoring. Results from all of these experiments are consistent with a role for GAs in the differential shoot growth that follows gravitropism, although the results do not eliminate the possibility that the redistribution of GAs results from the gravitropic response.     

Radioimmunoassay of 5-methoxy tryptophol in sheep plasma and pineal glands

No 5-methoxytryptophol (ML) could be detected in sheep plasma using a specific, sensitive radioimmunoassay developed for the purpose. Blood samples were collected from sheep during darkness and daylight and during various stages of the estrous cycle, but in no sample was the ML content above the detection limit of the method. Addition of 1 mM pargyline and neostigmine to blood immediately after collection to block metabolism did not result in a detectable ML concentration. The failure to detect ML was not due to degradation since added ML was not degraded by blood enzymes even after 16 hours incubation at 37 degrees C. Injection of 100 microgram and 1 mg ML sc in sheep resulted in a rapid rise of ML to 95-130pg/ml and 560-1000pg/ml respectively and disappearing with a half life of approximately 15-20 minutes. Sheep pineal glands collected during the light phase contained ML (51 +/- 5pg/mg tissue. X +/- SE, n = 7) which represents less than 6% of the melatonin content. It is concluded that if ML is present in sheep blood it is present at very low levels. It is thus unlikely to be a major circulating pineal hormone in this species, however, its role within the CNS as a local hormone cannot be excluded.     

New Methods of Chromatographic Separation of Gibberellins A1 and A3

Biologia Plantarum - New Chromatographic methods, chromatography in centrifugal field and thin-layer chromatography on alumina, were used for separating physiologically active gibberellins A1 and...     

Gibberellins and Heterosis in Maize : II. Response to Gibberellic Acid and Metabolism of [H]Gibberellin A(20)

Two maize inbreds, CM7 and CM49, and CM7 × CM49, their F₁ hybrid (which displayed significant heterosis), were examined with regard to response to exogenous gibberellin A₃ (GA₃), and in their ability to metabolize GA₂₀, a native GA of maize. The leaf sheath elongation response to GA₃ was far greater for the imbreds than for their hybrid. The inbreds also displayed significant elongation of the leaf blades in response to GA₃, whereas the hybrid was unaffected. Promotion of cell division in the leaf sheath of CM7 and the hybrid was effected by GA₃, but no promotion of cell elongation was observed in CM49, even though significant leaf sheath elongation occurred. Shoot dry weight of both inbreds was significantly increased by GA₃, but response by the hybrid in this parameter was slight and variable. Root dry weight of CM7 was significantly increased by GA₃, but was unchanged in CM49 and the hybrid. Thus, inbred shoot dry weight increases effected by GA₃ were not at the expense of the root system. Rapid metabolism of [2,3-³H]GA₂₀ occurred in all genotypes, although genotypic differences were observed. The hybrid had the highest rates of metabolism to GA glucosyl conjugate-like substances. Oxidative metabolism was also fastest in the hybrid, followed by CM7, and slowest in CM49, the slowest-growing inbred. Thus, rate of GA₂₀ metabolism is under genetic control in normal (i.e. not dwarfed) maize genotypes. These results, taken together with previous reports that the hybrid has significantly enhanced levels of endogenous GA-like substances, suggest that GA play a role in the expression of heterosis in maize.     

Gibberellins and Light-Stimulated Seed Germination

Bioactive gibberellins (GAs) promote seed germination in a number of plant species. In dicots, such as tomato and Arabidopsis, de novo GA biosynthesis after seed imbibition is essential for germination. Light is a crucial environmental cue determining seed germination in some species. The red (R) and far-red light photoreceptor phytochrome regulates GA biosynthesis in germinating lettuce and Arabidopsis seeds. This effect of light is, at least in part, targeted to mRNA abundance of GA 3-oxidase, which catalyzes the final biosynthetic step to produce bioactive GAs. The R-inducible GA 3-oxidase genes are predominantly expressed in the hypocotyl of Arabidopsis embryos. This predicted location of GA biosynthesis appears to correlate with the photosensitive site determined by using R micro-beam in lettuce seeds. The GA-deficient non-germinating mutants have been useful for studying how GA stimulates seed germination. In tomato, GA promotes the growth potential of the embryo and weakens the structures surrounding the embryo. Endo-b-mannanase, which is produced specifically in the micropylar endosperm in a GA-dependent manner, may be responsible for breaking down the endosperm cell walls to assist germination. Recently, a role for GA in overcoming the resistance imposed by the seed coat was also suggested in Arabidopsis from work with a range of seed coat mutants. Towards understanding the GA signaling pathway, GA response mutants have been isolated and characterized, some of which are affected in GA-stimulated seed germination.     

赤霉素与果树的生长发育

本文分析讨论了分子结构不同的赤霉素在生理作用上的差异,以及不同种类赤霉素对果树不同生长发育阶段的影响,介绍了与果树生长发育相关的赤霉素类型,并指出了今后在研究应用上须解决的问题。     

Gibberellins and pea seed development

The gibberellin (GA)-biosynthesis mutations, lh ⁱ , ls and Ie ⁵⁸³⁹ have been used to investigate the role(s) of the GAs in seed development of the garden pea (Pisum sativum L.). Seeds homozygous for lh ⁱ possess reduced GA levels, are more likely to abort during development, and weigh less at harvest, compared with wild-type seeds due to expression of the lh ⁱ mutation in the embryo and/ or endosperm. Compared with wild-type seeds, the lh ⁱ mutation reduces endogenous GA₁ and gibberellic acid (GA₃) levels in the embryo/endosperm a few days after anthesis and fertilizing lh ⁱ plants with wild-type pollen dramatically increases GA₁ and GA₃ levels in the embryo/ endosperm and restores normal seed development. By contrast, the ls and le ⁵⁸³⁹ mutations do not appear to reduce GA levels in the embryo/endosperm of seeds a few days after anthesis, and do not affect embryo or endosperm development. However, both the ls and lh ⁱ mutations substantially reduce endogenous GA levels in embryos at contact point (the first day the liquid endosperm disappears). Levels of GAs in seeds from crosses involving the ls and lh ⁱ mutations suggest that GAs are synthesised in both the embryo/endosperm and testa and that the expression of ls depends on the tissue and developmental stage examined. These results suggest that GAs (possibly GA₁ and/or GA₃) play an important role early in pea seed development by regulating the development of the embryo and/or endosperm. By contrast, the high GA levels found in wild-type seeds at contact point (and beyond) do not appear to have a physiological role in seed development.Abbreviations GA_n gibberellin A_n - DAA days after anthesis - WT wild-type We thank Noel Davies, Katherine McPherson and Peter Bobbi for technical assistance, Professor L. Mander (ANU, Canberra) for dideuterated GA standards, and the Australian Research Council and Frontier Research Program, The Institute of Physical and Chemical Research (RIKEN, Japan), for financial support.     

Radioimmunoassay of ochratoxin A in barley

A simple and rapid radioimmunoassay was developed for the quantitative determination of ochratoxin A in barley. [14C]ochratoxin A, with a specific activity of 130 Ci/mol, was used as the tracer. Toxin levels below 100 ng/ml required a cleanup step. Three methods (the Association of Official Analytical Chemists cleanup method, the solvent partition method, and the Extrelut 3 column cleanup method) were compared.     

Two New Gibberellins A50 and A52 in Seeds of Lagenaria leucantha

Two new gibberellins A₅₀ and A₅₂ were isolated from seeds of Lagenaria leucantha Rusby var. clavata Makino. Their structures were shown to be ent-2α,3α,10,llα-tetrahydroxy-20-norgibberell-16-ene-7,19-dioic acid 19,10-lactone (1) and ent-2α,3α,11β20-tetrahydroxy-gibberell-16-ene-7,19-dioic acid 19,20-lactone (10), respectively.