Transgenic herbicide-resistant Atropa belladonna using an Ri binary vector and inheritance of the transgenic trait

Summary Transgenic Atropa belladonna conferred with a herbicide-resistant trait was obtained by transformation with an Ri plasmid binary vector and plant regeneration from hairy roots. We made a chimeric construct, pARK5, containing the bar gene encoding phosphinothricin acetyltransferase flanked with the promoter for cauliflower mosaic virus 35S RNA and the 3 end of the nos gene. Leaf discs of A. belladonna were infected with Agrobacterium rhizogenes harboring an Ri plasmid, pRi15834, and pARK5. Transformed hairy roots resistant to bialaphos (5 mg/l) were selected and plantlets were regenerated. The integration of T-DNAs from pRi15834 and pARK5 were confirmed by DNA-blot hybridization. Expression of the bar gene in transformed R₀ tissues and in backcrossed F1 progeny with a nontransformant and self-fertilized progeny was indicated by enzymatic activity of the acetyltransferase. The transgenic plants showed resistance towards bialaphos and phosphinothricin. Tropane alkaloids of normal amounts were produced in the transformed regenerants. These results present a successful application of transformation with an Ri plasmid binary vector for conferring an agronomically useful trait to medicinal plants.Abbreviations CaMV cauliflower mosaic virus - NPT-II neomycin phosphotransferase II - PAT phosphinothricin acetyltransferase - PPT phosphinothricin     

Characteristics in tyrosine coordinations of four hemoglobins M probed by resonance Raman spectroscopy

Resonance Raman spectra of four hemoglobins (Hbs) M with tyrosinate ligand, that is, Hb M Saskatoon (beta distal His----Tyr), Hb M Hyde Park (beta proximal His----Tyr), Hb M Boston (alpha distal His----Tyr), and Hb M Iwate (alpha proximal His----Tyr), were investigated in order to elucidate structural origins for distinctly facile reducibility of the abnormal subunit of Hb M Saskatoon in comparison with other Hbs M. All of the Hbs M exhibited the fingerprint bands for the Fe-tyrosinate proteins around 1600, 1500, and 1270 cm-1. However, Hb M Saskatoon had the lowest Fe-tyrosinate stretching frequency and was the only one to display the Raman spectral pattern of a six-coordinate heme for the abnormal beta subunit; the others displayed the patterns of a five-coordinate heme. The absorption intensity of Hb M Saskatoon at 600 nm indicated a transition with a midpoint pH at 5.2, whereas that of Hb M Boston was independent of pH from 7.2 to 4.8. The fingerprint bands for the tyrosinate coordination as well as the Fe-tyrosinate stretching band disappeared for Hb M Saskatoon at pH 5.0, and the resultant Raman spectrum resembled that of metHb A, while those bands were clearly observed for Hb M Boston at pH 5.0 and for two Hbs M at pH 10.0. These observations suggest that the unusual characteristics of the heme in the abnormal beta chain of Hb M Saskatoon result from the weak Fe-tyrosinate bond, which allows weak coordination of the proximal histidine, giving rise to the six-coordinate high-spin state at pH 7.(ABSTRACT TRUNCATED AT 250 WORDS)     

Uptake, metabolism and distribution of nitrogen in crop plants traced by enriched and natural 15N: Progress over the last 30 years

The major findings of many years of research into plant N cycling are summarised in this review, firstly as revealed by ¹⁵N-enriched methods and secondly, in relation to natural ¹⁵N abundance (δ¹⁵N) in plants and their metabolites. This work has mainly been done in an agricultural context. As many groups especially attempt to relate δ¹⁵N to N cycling, atmospheric N deposition and the interactions of N with carbon budgets, we deem it useful to synthesize these major findings. Primary assimilation and distribution of N within plants were investigated from the ¹⁵N enrichment in individual plant organs and in individual amino acids after feeding them ¹⁵N-labelled compounds. In both roots and leaves, NH₄ ⁺ and NO₃ ^? were assimilated into amino acids, largely by a combination of glutamine synthetase (GS) and glutamate synthase (GOGAT). In the leaves, the transfer of glutamine (amide) N to glutamic acid was accelerated in the light, and amino N in some amino acids was deaminated to ammonia in the dark, followed by its incorporation into glutamine. The N in the growing parts such as growing leaves, filling grains and growing root parts were from two sources: re-allocation (phloem supply) of reserved N (amino acids), and currently-absorbed N. The metabolites from the mature parts may perform the roles of substrates for plant growth and signals for gene expression. δ¹⁵N values, measured for plants/soils and plant metabolites (inorganic N, amino acids, polyamines) were related with the acquisition, metabolism and distribution of N in plants. Small ¹⁵N/¹⁴N fractionation in the acquisition of N₂ and NO₃ ^? and large ¹⁵N/¹⁴N fractionation in NH₄ ⁺ uptake were found. The δ¹⁵N values of whole shoots or grains from field-grown crops were largely reflected major sources of N. In some legumes, ¹⁵N was enriched in their nodules and an extremely ¹⁵N-enriched compound was homospermidine. Nitrate reduction to ammonia (NR) and ammonia assimilation to glutamine (GS) showed large ¹⁵N/¹⁴N fractionations. Specific attention was paid to the δ¹⁵N values in xylem and phloem exudates compared to those of plant organs.     

An improved system for exposure of cultured mammalian cells to gaseous compounds in the chromosomal aberration assay

A gas exposure system using rotating vessels was improved for exposure of cultured mammalian cells to gaseous compounds in the chromosomal aberration assay. This system was composed of 12 square culture vessels, a device for preparation of air containing test gas, and positive and negative control gases at target concentrations and for supplying these gases to the culture vessels, and a roller apparatus in an incubator. Chinese hamster lung cells (CHL/IU) were grown on one side of the inner surface of the square culture vessel in the MEM medium. Immediately prior to exposure, the medium was changed to the modified MEM. Air in the culture vessel was replaced with air containing test gas, positive or negative control gas. Then, the culture vessels were rotated at 1.0 rpm. The monolayered culture cells were exposed to test gas during about 3/4 rotation at upper positions and alternatively immersed into the culture medium during about 1/4 rotation at lower positions. This system allowed the chromosomal aberration assay simultaneously at least at three different concentrations of a test gas together with positive and negative control gases with and without metabolic activations, and duplicate culture at each exposure concentration. Seven gaseous compounds, 1,3-butadiene, chlorodifluoromethane, ethyl chloride, methyl bromide, methyl chloride, propyne, and vinyl chloride, none of which has been tested to date, were tested on CHL/IU for the chromosomal aberration assay using this gas exposure system. All the compounds except chlorodifluoromethane showed positive responses of the structural chromosomal aberrations, whereas polyploidy was not induced by any of these gases. This improved gas exposure system proved to be useful for detecting chromosomal aberrations of gaseous compounds.     

Enhanced expression of RNase L as a novel intracellular signal generated by NMDA receptors in mouse cortical neurons

Recently we showed that the level of mitochondrial mRNA was decreased prior to neuronal death induced by glutamate. As the level of mRNA is regulated by ribonuclease (RNase), we examined RNase activity and its expression in the primary cultures of cortical neurons after glutamate treatment in order to evaluate the involvement of RNase in glutamate-induced neuronal death. A 15-min exposure of the cultures to glutamate at the concentration of 100muM produced marked neuronal damage (more than 70% of total cells) at 24-h post-exposure. Under the experimental conditions used, RNA degradation was definitely observed at a period of 4-12-h post-exposure, a time when no damage was seen in the neurons. Glutamate-induced RNA degradation was completely prevented by the N-methyl-d-aspartic acid (NMDA) receptor channel blocker MK-801 or the NR2B-containing NMDA receptor antagonist ifenprodil. Glutamate exposure produced enhanced expression of RNase L at least 2-12h later, which was absolutely abolished by MK-801. However, no significant change was seen in the level of RNase H1 mRNA at any time point post-glutamate treatment. Immunocytochemical studies revealed that RNase L expressed in response to glutamate was localized within the nucleus, mitochondria, and cytoplasm in the neurons. Taken together, our data suggest that expression of RNase L is a signal generated by NMDA receptor in cortical neurons. RNase L expression and RNA degradation may be events that cause neuronal damage induced by NMDA receptor activation.     

Effect of nitrogen-donor ancillary ligand on oxidation behavior of Co(III) binuclear complexes with conjugated bis(catecholate) ligands

Two binuclear complexes of cobalt(III) have been prepared with 3,3′,4,4′-tetrahydroxy-5,5′-di-tert-butylbenzaldazine (H₄thBu) as bis(catecholate) ligand and two different ancillary ligands, 2,2′-bipyridine (bpy) or 2,2′-dipyridylamine (dpa). These compounds were characterized by ¹H NMR spectra, electrochemical measurements and UV–Vis spectra. In one case, [Co₂(dpa)₄(thBu)]²⁺, electrochemical oxidation of the complexes occurs at the bridges as two closely spaced one-electron couples (E_1/2 = 1 mV and 168 mV versus Fc/Fc⁺). Chemical oxidation of [Co₂(dpa)₄(thBu)]²⁺ using Ag⁺ is observed to occur as a stepwise two-electron process forming [Co₂(dpa)₄(thBu^Cat,SQ)]³⁺ or [Co₂(dpa)₄(thBu^SQ,SQ)]⁴⁺ by UV–Vis spectrum. However, [Co₂(bpy)₄(thBu)]²⁺ shows no change in electronic spectrum under the same conditions of oxidation. This illustrates the dependence of redox properties of the binuclear Co(III) complexes on the nature of the nitrogen-donor ancillary ligands. In this report we discuss the effect of two different nitrogen-donor ancillary ligands on the0 oxidation behavior of binuclear Co(III) complexes.