Successful expression in pollen of various plant species of in vitro synthesized mRNA introduced by particle bombardment

Gold particles coated with -glucuronidase (GUS) mRNA with a 5 cap structure that had been synthesized in vitro were introduced, by use of a pneumatic particle gun, into pollen grains of lily (Lilium longiflorum), freesia (Freesia refracta) and tulip (Tulipa gesneriana). A fluorometric assay for the GUS activity indicated that in vitro synthesized GUS mRNA introduced into these pollen cells by particle bombardment was successfully expressed. GUS activity in extracts of the bombarded lily pollen became detectable fluorometrically within 30 min after bombardment, peaked at 6 h, then gradually decreased. This activity changed as a function of the developmental stage of the pollen cell of lily.     

Frequency-dependent interference by magnetic fields of nerve growth factor-induced neurite outgrowth in PC-12 cells

We have shown that 50 Hz sinusoidal magnetic fields within the 5-10 micro Tesla (μT) rms range cause an intensity-dependent reduction in nerve growth factor (NGF) stimulation of neurite outgrowth (NO) in PC-12 cells. Here we report on the frequency dependence of this response over the 15-70 Hz range at 5 Hz intervals. Primed PC-12 cells were plated in collagen-coated, 60 mm plastic petri dishes with or without 5 ng/ml NGF and were exposed to sinusoidal magnetic fields for 22 h in a CO₂ incubator at 37 °C. One 1,000-turn coil, 20 cm in diameter, generated vertically oriented magnetic fields. The dishes were stacked on the center axis of the coil to provide a range of intensities between 3.5 and 9.0 μT rms. The flux density of the ambient DC magnetic field was 37 μT vertical and 19 μT horizontal. The assay consisted of counting over 100 cells in the central portion (radius ≤0.3 cm) of each dish and scoring cells positive for NO. Sham exposure of cells treated identically with NGF demonstrated no difference in the percentage of cells with NO between exposed and magnetically shielded locations within the incubator. Analysis of variance demonstrated flux density-dependent reductions in NGF-stimulated NO over the 35-70 Hz frequency range, whereas frequencies between 15 Hz and 30 Hz produced no obvious reduction. The results also demonstrated a relative maximal sensitivity of cells at 40 Hz with a possible additional sensitivity region at or above 70 Hz. These findings suggest a biological influence of perpendicular AC/DC magnetic fields different from those identified by the ion parametric resonance model, which uses strictly parallel AC/DC fields. © 1995 Wiley-Liss, Inc.     

Studies of mercurated derivatives of acetylacetone and ethyl acetoacetate by solid state nuclear magnetic resonance and vibrational spectroscopies

Seven new mono- and/or dimercurated compounds involving acetylacetone (2,4-pentanedione) or ethyl acetoacetate (3-ethyl ketobutanoate) were synthesized in aqueous medium. In all cases, mercuration occurred at methylene carbon atoms. All compounds were carefully analyzed by solid state carbon-13 nuclear magnetic resonance. Assignments were confirmed by using selective sequences, which allowed a total editing of the spectra. It was shown that deshielding of ¹³C mercurated sites occurred when the rate of mercuration increased. It was also possible to measure direct ¹J(¹⁹⁹Hg¹³C) coupling constants. The main bands of the vibrational spectra (infrared and Raman) were assigned. It was proved that v(C = O) and δ(C(γ)-H) could be directly related to the mercuration rate of molecules.     

Preparation of immobilized enzyme with high activity using affinity tag based on proteins A and G

Affinity tag AG consisting of immunoglobulin G (lgG)-binding domains of protein A from Staphylococcus aureus (EDABC) and those of protein G from Streptococcus strain G148 (C2C3) were used to facilitate immobilization of beta-galactosidase (betagal) from Escherichia coli. Poly(methylmethacrylate/N-isopropylacrylamide/methacrylic acid) [P(MMA/NIPAM/MAA)] and poly(styrene/N-isopropylacrylamide/methacrylic acid) [P(St/NIPAM/MAA)] latex particles, which show thermosensitivity, were used as support materals to prepare affinity adsorbents. Human gamma-globulin (HgammaGb), whose major fraction is lgG, was used as an affinity ligand and was covalently immobilized onto the both latex particles by the carbodiimide method under various conditions. A fusion protein, AGbetagal, was immobilized at pH 7.3 by the specific binding of affinity tag to these affinity adsorbents. The amount of adsorbed AGbetagal per unit amount of immobilized HgammaGb, namely, efficiency of ligand utilization, was strongly affected by the type of latex particles and pH value for HgammaGb immobilization. The efficiency of ligand utilization was maximum in the affinity adsorbents prepared at pH 6.0 to 7.0, and that in the HgammaGb-P(MMA/NIPAM/MAA) latex particles was high. This result could be explained by the conformation and orientation of immobilized HgammaGb molecules. Immobilized AGbetagal retained approximately 75% of its activity in solution and the binding is stable enough to allow repeated use. These results clearly demonstrate that combination of the affinity tag AG and the affinity adsorbents, based on the thermosensitive latex particles, offers a simple and widely applicable method for preparation of immobilized enzyme with high activity. (c) 1995 John Wiley & Sons, Inc.     

Structural and immunochemical identification of Leb glycolipids in the plasma of a group O Le(a-b-) secretor

Total non-acid glycosphingolipids were isolated from the plasma of a healthy red blood cell group O Le(a-b-) salivary ABH secretor individual. Glycolipids were fractionated by HPLC and combined into eight fractions based on chromatographic and immunoreactive properties. These glycolipid fractions were analysed by thin-layer chromatography and tested for Lewis activity with antibodies reactive to the type 1 precursor (Le^c), H type 1 (Le^d), Le^a and Le^b epitopes. Fractions were structurally characterized by mass spectrometry (EI-MS and LSIMS) and proton NMR spectroscopy. Expected blood group glycolipids, such as H type 1, (Fuc1-2Gal1-3GlcNAc1-3Gal1-4Glc1-1Cer) were immunochemically and structurally identified. Inconsistent with the red cell phenotype and for the first time, small quantities of Le^b blood group glycolipids (Fuc1-2Gal1-3(Fuc1-4)GlcNAc1-3Gal1-4Glc1-1Cer) were immunochemically and structurally identified in the plasma of a Lewis-negative individual. These findings confirm recent immunological evidence suggesting the production of small amounts of Lewis antigens by Lewis negative individuals. Abbreviations: HPLC, high performance liquid chromatography; TLC, (high performance) thin layer chromatography; EI-MS, electron impact ionisation mass spectrometry; LSIMS, liquid secondary ion mass spectrometry; NMR, nuclear magnetic resonance spectroscopy. The sugar types are abbreviated to Hex for hexose, HexNAc forN-acetylhexosamine and dHex for deoxyhexose (fucose). The ceramide types are abbreviated to d for dihydroxy and t for trihydroxy base, n for non-hydroxy and h for hydroxy fatty acids; LCB, long chain base.     

Detection and minimization of H-phosphonate side reaction during phosphopeptide synthesis by a post-assembly global phosphorylation strategy

Summary In the course of solid-phase synthesis of phosphopeptides by a post-assembly global phosphorylation strategy, the corresponding H-phosphonate peptides form as byproducts. We describe model studies to investigate this side reaction as a function of reaction conditions, and use this information to develop conditions that minimize the problem, i.e., use of dibenzyl N,N-di-isopropyl phosphoramidite for phosphitylation, followed immediately by oxidation with anhydrous tert-butyl hydroperoxide in dry tetrahydrofuran under argon, and final acidolytic cleavage.This work was taken in part from the Ph.D. Theses of E.A. Ottinger (1994) and Q. Xu (1996), University of Minnesota, Minneapolis, MN, U.S.A. Preliminary presentations of portions of this work were made at the Twenty-Second European Peptide Symposium, Interlaken, Switzerland, September 13–19, 1992, see Ref. 1, at the 14th American Peptide Symposium, Columbus, OH, U.S.A., June 18–23, 1995, and at the Fourth International Symposium on Solid Phase Synthesis & Combinatorial Chemical Libraries, Edinburgh, Scotland, U.K., September 12–16, 1995, see Ref. 2. The title side reaction was first discussed for tyrosine (see Refs 1 and 3), but all of the mechanism studies discussed herein are for serine and threonine.Amino acid symbols denote the l-configuration, and abbreviations for amino acids and peptides follow rules of the IUPAC-IUB Commission of Biochemical Nomenclature [J. Biol. Chem., 247 (1972) 977].     

Complete 1H, 13C, and 15N NMR resonance assignments and secondary structure of human glutaredoxin in the fully reduced form.

Human glutaredoxin is a member of the glutaredoxin family, which is characterized by a glutathione binding site and a redox-active dithiol/disulfide in the active site. Unlike Escherichia coli glutaredoxin-1, this protein has additional cysteine residues that have been suggested to play a regulatory role in its activity. Human glutaredoxin (106 amino acid residues, M(r) = 12,000) has been purified from a pET expression vector with both uniform 15N labeling and 13C/15N double labeling. The combination of three-dimensional 15N-edited TOCSY, 15N-edited NOESY, HNCA, HN(CO)CA, and gradient sensitivity-enhanced HNCACB and HNCO spectra were used to obtain sequential assignments for residues 2-106 of the protein. The gradient-enhanced version of the HCCH-TOCSY pulse sequence and HCCH-COSY were used to obtain side chain 1H and 13C assignments. The secondary structural elements in the reduced protein were identified based on NOE information, amide proton exchange data, and chemical shift index data. Human glutaredoxin contains five helices extending approximately from residues 4-10, 24-36, 53-64, 83-92, and 94-104. The secondary structure also shows four beta-strands comprised of residues 15-19, 43-48, 71-75, 78-80, which form a beta-sheet almost identical to that found in E. coli glutaredoxin-1. Complete 1H, 13C, and 15N assignments and the secondary structure of fully reduced human glutaredoxin are presented. Comparison to the structures of other glutaredoxins is presented and differences in the secondary structure elements are discussed.     

Solid-state fermentation of carob pods byAspergillus niger for protein production: effect of particle size

Two strains ofAspergillus niger were cultured in solid-state fermentation system on carob pods ground from 1.25 to 8 mm diam. A particle size of 2.5 mm gave the highest protein content of the final product (20%, w/w) and 52% of the total soluble carbohydrates were utilized. The total tannin concentration of the carob pods decreased by 83% in 4 days of fermentation.T. Smail and O. Salhi are with the Laboratory of Microbiology, U.R.B.A.F., Institute of Biology, Tizi-Ouzou University, Algeria. J.S. Knapp is with the Department of Microbiology, The University of Leeds, Leeds LS2 9JT, UK;     

Development of a pollen-mediated transformation method forNicotiana glutinosa

The development is described of a new procedure to genetically transform plant species using the male gametophyte as a natural transformation vector. Our system avoids the need for complicated regeneration procedures thus making it broadly applicable. Naked plasmid DNA encoding kanamycin resistance and GUS activity was introduced by particle gun bombardment into mature pollen grains ofNicotiana glutinosa. Bombarded pollen was used for pollinations and the resulting seeds were selected for kanamycin resistance. Two different kanamycin-resistant plants, designated VIP A and VIP B, were obtained in two independent experiments. In VIP A, TR2-driven GUS activity was observed in vascular bundles, trichomes and in a small number of pollen grains. DNA gel blot analysis indicated that the introduced DNA was integrated independently into the genome of VIP A and VIP B. It was shown that male and female gametophyte development and seed set were highly aberrant in both VIP A and VIP B and that the offspring of self- and cross-pollinations did not contain the transgenes. This might be caused by a recombination event during the integration of the naked DNA resulting in a deletion of part of the target chromosome. After meiosis such a deletion is lethal for the gametes. Our observation that the transgenes were detected in DNA isolated from sporophytic tissues but not in DNA from VIP A and VIP B pollen grains is in line with this explanation. Future experiments designed to increase the frequency of transformation and to transfer the transgenes to the offspring are discussed.     

Magnetic field dependence of radical-pair decay kinetics and molecular triplet quantum yield in quinone-depleted reaction centers

Radical-pair decay kinetics and molecular triplet quantum yields at various magnetic fields are reported for quinone-depleted reaction centers from the photosynthetic bacterium Rhodopseudomonas sphaeroides R26. The radical-pair decay is observed by picosecond absorption spectroscopy to be a single exponential to within the experimental uncertainty at all fields. The decay time increases from 13 ns at zero field to 17 ns at 1 kG, and decreases to 9 ns at 50 kG. The orientation averaged quantum yield of formation of the molecular triplet of the primary electron donor, ³P, drops to 47% of its zero-field value at 1 kG and rises to 126% at 50 kG. Combined analysis of these data gives a singlet radical-pair decay rate constant of 5 · 10⁷s^?1, a lower limit for the triplet radical-pair decay rate constant of 1 · 10⁸s^?1 and a lower limit for the quantum yield of radical-pair decay by the triplet channel of 38% at zero field. The upper limit of the quantum yield of ³P formation at zero field is measured to be 32%. In order to explain this apparent discrepancy, decay of the radical pair by the triplet channel must lead to some rapid ground state formation as well as some ³P formation. It is proposed that the triplet radical pair decays to a triplet charge-transfer state which is strongly coupled to the ground state by spin-orbit interactions. Several possibilities for this charge-transfer state are discussed.