Metabolism of gibberellin a(12)-7-aldehyde by soybean cotyledons and its use in identifying gibberellin a(7) as an endogenous gibberellin

The level of gibberellin(GA)-like material in cotyledons of soybean (Glycine max L.) was highest at mid-pod fill—about 10 nanograms GA₃ equivalents per gram fresh weight of tissue, assayed in the immersion dwarf rice bioassay. This amount is about 1000-fold less than levels in Pisum and Phaseolus seed, other legume species whose spectrum of endogenous gibberellins (GAs) is well known. The metabolism of [¹⁴C]-GA₁₂-7-aldehyde (GA₁₂ald)—the universal GA precursor—by intact, mid-pod-fill, soybean cotyledons and their cell-free extracts was investigated. In 4 hours, extracts converted GA₁₂ald to two products—[¹⁴C]GA₁₂ (42% yield) and [¹⁴C]GA₁₅ (7%). Within 5 minutes, intact embryos converted GA₁₂ald to [¹⁴C]GA₁₂ and [¹⁴C]GA₁₅ in 15% yield; 4 hour incubations afforded at least 22 products (96% total yield). The putative [¹⁴C]GA₁₂ was identified as a product of [¹⁴C]GA₁₂ald metabolism on the basis of co-chromatography with authentic GA₁₂ on a series of reversed and normal phase high pressure liquid chromatography (HPLC) and thin-layer chromatography (TLC) systems, and by a dual feed of the putative [¹⁴C]GA₁₂ and authentic [¹⁴C]GA₁₂ to cotyledons of both peas and soybeans. The [¹⁴C]GA₁₅ was identified as a metabolite of [¹⁴C]GA₁₂ald by capillary gas chromatography (GC)-mass-spectrometry-selected ion monitoring, GC-radiocounting, HPLC, and TLC. By adding the [¹⁴C] metabolites of [¹⁴C]GA₁₂ald to a different and larger extract (about 0.2 kg fresh weight of soybean reproductive tissue) and purifying endogenous substances co-chromatographing with these metabolites, at least two GA-like substances were obtained and one identified as GA₇ by GC-mass spectrometry. Since [¹⁴C]GA₉ was not found as a [¹⁴C]metabolite of [¹⁴C]GA₁₂ald, soybean embryos might have a pathway for biosynthesis of active, C-19 gibberellins like that of the cucurbits; GA₁₂ald → GA₁₂ → GA₁₅ → GA₂₄ → GA₃₆ → GA₄ → GA₇.     

Transformation of Arthrobacter and studies on the transcription of the Arthrobacter ermA gene in Streptomyces lividans and Escherichia coli.

We report the development of a plasmid-mediated transformation system for Arthrobacter sp. NRRLB3381, using the Streptomyces cloning vector pIJ702. Our procedure gives a transformation frequency of 10(3)/micrograms of plasmid DNA. In addition we have explored the expression of the Arthrobacter ermA gene in Streptomyces lividans and Escherichia coli, and shown that the ermA promoter is recognized in S. lividans not E. coli. The relationship between Arthrobacter, Streptomyces and E. coli promoters is discussed.     

Effect of shading individual soybean reproductive structures on their abscisic Acid content, metabolism, and partitioning

Pod set in soybean is related to carbon partitioning and may be, at least partially, regulated by abscisic acid (ABA) concentrations. The studies reported here examine the relationship between carbon and ABA partitioning, reproductive abscission and ABA metabolism. The partitioning of radiolabeled ABA and photoassimilates from leaves to flowers and endogenous ABA concentrations were determined in shaded and unshaded reproductive structures. Aluminum foil was gently placed over individual soybean reproductive structures for 48 hours at 0, 4, 12, 17, and 22 days after anthesis (DAA). Shading of flowers at 12, 17, and 22 DAA resulted in significantly reduced concentration of ABA. However, shading had no effect on the catabolism of exogenously supplied [³H] ABA. The shading treatment on the first four of the five dates reduced partitioning of photoassimilates and ABA from the subtending leaf to the flower. Shading of reproductive structures also caused a significant reduction in the amount of assimilate exported from the subtending leaf, at 17 DAA. We conclude that shade-induced premature reproductive abscission in soybean is not stimulated by high levels of ABA within reproductive structures, but that ABA may inhibit abscission of reproductive structures by playing a role in preferential assimilate partitioning.     

Distribution of Abscisic Acid in Maize Kernel during Grain Filling

The distribution of abscisic acid (ABA) within maize (Zea mays L.) kernels was studied in kernels from nontreated plants, from plants in which assimilate supply had been altered by source/sink manipulations, and in kernels cultured in vitro on ABA-free media. Prior to growth of the embryo, both the pedicel/placento-chalazal complex and the endosperm contained high concentration of ABA; however, the quantity of ABA in these tissues declined as the concentration in the embryo increased during the early stages of embryo growth. Peaks in the levels of ABA appeared to occur prior to and not concurrent with physiological events during grain filling. During most of the grain filling period, ABA concentration in the embryo was higher than that found in other kernel components. Altering assimilate supply by partial defoliation at two stages of development resulted in variable and transient effects on the relative distribution and concentration of ABA in kernel components. The concentration and distribution of ABA among components of kernels grown in vitro was similar to that observed for field-grown kernels. On the basis of these findings, in situ synthesis of ABA by kernel components is implicated and the putative role of ABA in the regulation of kernel development is discussed.     

Pentobarbital Antagonizes the A1 Adenosine Receptor-Mediated Inhibition of Hippocampal Neurotransmitter Release

Barbiturates have been shown to be competitive antagonists at A1 adenosine receptors in radioligand binding studies. The present study investigates the effects of pentobarbital on the A1 receptor-mediated inhibition of neurotransmitter release from rabbit hippocampal slices. The inhibition of the electrically evoked release of [3H]noradrenaline by the A1 receptor agonist (R)-N6-phenylisopropyladenosine (R-PIA) was antagonized by pentobarbital with an apparent pA2 value of 3.5. Low concentrations of pentobarbital alone altered neither basal nor evoked release of [3H]noradrenaline, whereas 1,000 microM pentobarbital enhanced the basal and reduced the evoked release. In the presence of 8-phenyltheophylline, pentobarbital (200 microM and 1,000 microM) reduced the evoked noradrenaline release. Pentobarbital also antagonized the inhibition of [3H]acetylcholine release by R-PIA. In contrast to the noradrenaline release model, the evoked release of acetylcholine was enhanced by the presence of pentobarbital (50-500 microM), an effect that was lost in the presence of 8-phenyltheophylline. These results indicate that pentobarbital, in addition to a direct inhibitory action at higher concentrations, has a facilitatory effect on neurotransmitter release by blocking presynaptic A1 adenosine receptors. The possible relevance of these findings for the excitatory effects of barbiturates is discussed.     

Lidocaine pharmacokinetics during water immersion in normal humans

Water immersion produces a marked diuresis, natriuresis, and kaliuresis in association with suppression of the renin-aldosterone system. These effects are mediated primarily by an increase in central blood volume. Consequently, this redistribution and the resultant marked increase in cardiac output is associated with alterations in the circulating levels of several volume regulatory hormones, including plasma renin activity and plasma aldosterone. Although the changes in these blood hormonal levels probably reflect perturbation of hormonal release, it is conceivable that the above-mentioned central hemodynamic modifications result in an altered splanchnic blood flow, thereby modulating hormonal clearances. We assessed the effects of immersion on hepatic blood flow by determining the pharmacokinetics of single doses of lidocaine administered intravenously. Seven normal male subjects were studied during a time-control period and during water immersion to the neck. The clearance of lidocaine was unaltered by immersion, suggesting that the presumed marked central hypervolemia and increased cardiac output was not associated with changes in splanchnic blood flow.     

Paramyosin gene (unc-15) of Caenorhabditis elegans. Molecular cloning, nucleotide sequence and models for thick filament structure

Paramyosin is a major structural component of thick filaments isolated from many invertebrate muscles. The Caenorhabditis elegans paramyosin gene (unc-15) was identified by screening with specific antibodies an "exon-expression" library containing lacZ/nematode gene fusions. Short probes recovered from the library were used to identify bacteriophage lambda and cosmid clones that encompass the entire paramyosin (unc-15) gene. From these clones, numerous subclones containing epitopes reacting with anti-paramyosin sera were obtained, providing strong evidence that the initial cloned fragment was, in fact, derived from the structural gene for paramyosin. The complete nucleotide sequence of a 12 x 10(3) base-pair region spanning the gene was obtained. The gene is composed of ten short exons encoding a protein of 866 [corrected] amino acid residues. Paramyosin is highly similar to residues 267 to 1089 of myosin heavy chain rods. For most of its length, paramyosin appears to form an alpha-helical coiled-coil and shows the expected heptad repeat of hydrophobic amino acid residues and the 28-residue repeat of charged amino acids characteristic of myosin heavy chain rods. However, paramyosin differs from myosin in having non-helical extensions at both the N and C termini and an additional "skip" residue that interrupts the 28-residue repeat. The distribution of charges along the length of the paramyosin rod is also significantly different from that of myosin heavy chain rods. Potential charge-mediated interactions between paramyosin rods and between paramyosin and myosin rods were calculated using a model successfully applied previously to the analysis of the myosin rod sequences. Myosin rods aligned in parallel show optimal charge-charge interactions at multiples of 98 residue staggers (i.e. at axial displacements of multiples of 143 A). Paramyosin rods, in contrast, appear to interact optimally at parallel staggers of 493 residues (i.e. at axial displacements of 720 A) but show only weak interaction peaks at 98 or 296 residues. Similar calculations suggest optimal interactions between paramyosin molecules and myosin rods and in their anti-parallel alignments. The implications of these results for the structure of the bare zone and the assembly of nematode thick filaments are discussed.