Interaction of a rhizobial DNA-binding protein with the promoter region of a plant leghemoglobin gene.

A nucleotide sequence was identified approximately 650 bp upstream of the Sesbania rostrata leghemoglobin gene Srglb3 start codon, which interacts specifically with a proteinaceous DNA-binding factor found in nodule extracts but not in extracts from leaves or roots. The binding site for this factor was delimited using footprinting techniques. The DNA-binding activity of this factor was found to be heat stable, dependent on divalent cations, and derived from the (infecting) Azorhizobium caulinodans bacteria or bacteroids (A. caulinodans bacterial binding factor 1, AcBBF1). A 9- to 10-kD protein was isolated from a free-living culture of A. caulinodans that co-purifies with the DNA-binding activity (A. caulinodans bacterial binding protein 1, AcBBP1) and interacts specifically with its target (S. rostrata bacterial binding site 1, SrBBS1). The amino acid sequence of the N-terminal 27 residues of AcBBP1 was determined and was found to share significant similarity (46% identity; 68% similarity) with a domain of the herpes simplex virus major DNA-binding protein infected cell protein 8 (ICP8). An insertion mutation in the SrBBS1 was found to result in a substantial reduction of the expression of a Srglb3-gus reporter gene fusion in nodules of transgenic Lotus corniculatus plants, suggesting a role for this element in Srglb3 promoter activity. Based on these results, we propose that (a) bacterial transacting factor(s) may play a role in infected cell-specific expression of the symbiotically induced plant lb genes.     

Investigation of the ‘double cross’ splitting mechanism of single-crystal diamond under nanoindentation via molecular dynamics simulation

Elucidating the mechanical response of diamond is a difficult task due to its ultrahard nature. Here, we applied a molecular dynamics (MD) method to investigate the mechanical response of single-crystal diamond under nanoindentation. There was no obvious “pop in” phenomenon on the load–depth curve, and the elastic modulus deduced from the curve was 1128 GPa, which was similar to the value obtained from experimental measurements. Results from computed tomography (CT) and the coordination number showed that the distribution of the mismatched C atoms around the deformation zone took the form of a ‘double cross.’ The atoms around the indenter tip could be divided into two zones, a translation zone and a lattice distortion zone, based on their movements. Subsequent first-principles calculations revealed that the C-atom displacement barrier varied significantly with direction, which resulted in shear stress between the two zones and the formation of the double-cross splitting.

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Graphical Abstract The displacement of the atoms around the indenter tip

     

Processing of proenkephalin-A in bovine chromaffin cells. Identification of natural derived fragments by N-terminal sequencing and matrix-assisted laser desorption ionization-time of flight mass spectrometry

A large variety of proenkephalin-A-derived peptides (PEAPs) are present in bovine adrenal medulla secretory granules that are cosecreted with catecholamines upon stimulation of chromaffin cells. In the present paper, after reverse phase high performance liquid chromatography of intragranular soluble material, PEAPs were immunodetected with antisera raised against specific proenkephalin-A (PEA) sequences (PEA63-70 and PEA224-237) and analyzed by matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) mass spectrometry. Thirty PEAPs were characterized in addition to enkephalins and whole PEA, indicating that preferential proteolytic attacks occurred at both N- and C-terminal regions. A similar approach was used to characterize PEA-derived fragments exocytotically released into the extracellular space that showed five additional minor PEAPs. Among all these naturally generated peptides, enkelytin, the antibacterial bisphos- phorylated C-terminal peptide (PEA209-237), was predominantly generated, as shown by MALDI-TOF mass spectrometry analysis, which constituted an efficient method for its identification. Finally, the data on PEA intragranular and extracellular processing in adrenal medulla are discussed in regard to the known enzymatic processing mechanisms. We note the high conservation of the cleavage points in evolutionarily diverse organisms, highlighting an important biological function for the released PEAPs.     

Mono-unsaturated fatty acids protect against beta-cell apoptosis induced by saturated fatty acids, serum withdrawal or cytokine exposure

Long-chain saturated fatty acids are cytotoxic to pancreatic β-cells while shorter-chain saturated and long-chain unsaturated molecules are better tolerated. Mono-unsaturated fatty acids are not, however, inert since they inhibit the pro-apoptotic effects of saturated molecules. In the present work we show that the mono-unsaturates palmitoleate (C16:1) or oleate (C18:1) also cause marked inhibition of apoptosis induced by exposure of clonal BRIN-BD11 β-cells to serum withdrawal or a combination of interleukin-1β plus interferon-γ. This response was dose-dependent and not accompanied by changes in NO formation. Taken together, the results suggest that mono-unsaturated fatty acids regulate a distal step common to several apoptotic pathways in pancreatic β-cells.     

Alterations in neutrophil (PMN) free intracellular alpha-keto acid profiles and immune functions induced by L-alanyl-L-glutamine, arginine or taurine

Summary. The objective of this study was to determine the dose as well as duration of exposure-dependent effects of L-alanyl-L-glutamine, arginine or taurine on polymorphonuclear neutrophil (PMN) free α-keto acid profiles and, in a parallel study, on PMN immune functions. Exogenous L-alanyl-L-glutamine significantly increased PMN α-ketoglutarate, pyruvate PMN superoxide anion (O₂⁻) generation, hydrogen peroxide (H₂O₂) formation and released myeloperoxidase (MPO) activity. Arginine also led to significant increases in α-ketoglutarate, pyruvate, MPO release and H₂O₂ generation. Formation of O₂⁻ on the other hand was decreased by arginine. Incubation with taurine resulted in lower intracellular pyruvate and α-ketobutyrate levels, decreased O₂⁻ and H₂O₂ formation and a concomitant significantly increased MPO activity. We therefore believe that considerable changes in PMN free-α-keto-acid profiles, induced for example by L-alanyl-L-glutamine, arginine or taurine, may be one of the determinants in cell nutrition that considerably modulates the immunological competence of PMN.     

Chromosome substitutions and recombination in the amphiploid Lolium perenne×Festuca pratensis cv Prior (2n=4x=28)

 The synthetic amphiploid cv Prior was created in the early 1970s at the Welsh Plant Breeding Station by crossing colchicine-induced autotetraploids of Lolium perenne (2n=14) and Festuca pratensis (2n=14). Meiosis in the early generations was characterized as stable, with frequent bivalent formation. In situ hybridization of a L. perenne total genomic DNA probe to mitotic chromosome spreads of 12 plants, from two extant populations of Prior, demonstrates extensive recombination between the two genomes. Recombination events occur along the whole length of chromosome arms but with a higher frequency in the medial portion. The species origins of chromosomes were assigned by the presence or absence of a fluorescent probe at the centromere. There has been a substitution of Festuca-origin chromosomes by those of Lolium-origin, resulting in a mean of 17.9 (15–21) Lolium and 9.7 (7–13) Festuca chromosomes per genotype. Mean chromatin length per genotype comprised 62.1% Lolium and 37.9% Festuca. On average 9.3 Lolium (51.1% of those present) and 3.5 Festuca (37.8%) chromosomes had no recombined segments. For chromosomes which did show recombination, fewer alien segments were observed in Lolium than in Festuca chromosomes. Festuca chromosomes in genotypes selected for drought resistance had undergone more recombination than in genotypes from an unselected population, though this difference was not statistically significant for the small sample examined. Received: 16 June 1998 / Accepted: 17 September 1998 RID="1" ID="1" <E5>Present address:</E5> Lithuanian Institute of Agriculture, 5051 Dotnuva-Akademija, Kedainiai, Lithuania RID=" ID=" Communicated by J. W. Snape RID=" ID=" <E5>Correspondence to</E5> P. H. Canter