The purification and immunocharacterisation of N-methylputrescine oxidase from transformed root cultures of Nicotiana tabacum L. cv SC58

The enzyme N-methylputrescine oxidase which catalyses the conversion of N-methylputrescine to N-methylpyrrolinium salt has been purified to homogeneity from transformed roots of Nicotiana tabacum L. cv SC58. The enzyme has an apparent sub-unit molecular weight of 53 kDa by sodium dodecyl sulphate-polyacrylamide gel electrophoresis with gel-filtration studies, indicating that the native form is a dimer. The K _m of the enzyme for N-methylputrescine has been estimated to be 0.1 mM. Polyclonal antibodies raised to the purified protein recognise one product in an immunoblot of a crude extract of transformed root tissue and will immunoprecipitate N-methylputrescine oxidase activity from such an extract. The antibodies also show a high degree of specificity in immunoblots of crude extracts of transformed root cultures from a range of other solanaceous and non-solanaceous species but do not cross-react with a partially purified preparation of pea-seedling diamine oxidase.Abbreviations MPO N-methylputrescine oxidase - PVDF polyvinylidene difluoride - SDS-PAGE sodium dodecyl sulphate-polyacrylamide gel electrophoresis We would like to thank members of the Plant Cell Biotechnology Group, Institute of Food Research, Norwich Laboratory, for their helpful discussions during the preparation of this paper.     

A matter of timing: System requirements for repair and their temporal dimensions

Research into repair within the circular economy (CE) typically focuses on technical aspects of design, policy, and markets, and often assumes simplified conditions for the user/owner and the product system to explain the barriers to scaling repair activities. However, factors occurring at pre-use stages of the product's life cycle can significantly influence whether, and to what extent, repair is viable or possible, that is, warranty duration, after-sale service provision, and access to necessities. The passing of time can directly and indirectly affect the ability, difficulty, and thus, the likelihood of repair activities being performed at each stage of the product's life cycle. Drawing from the literature and applying inductive systems-thinking tools, we propose a framework for considering the “System of Repairability.” We delineate how the passing of time (temporal dimensions) affects one's ‘‘ability to repair,’’ as a product progresses through different life cycle phases (i.e., breakdown vs. repair vs. disposal), and the point(s) at which the repair is considered or attempted (i.e., year of usage). By integrating life cycle and temporal (time-based) dimensions into a broad System of Repairability framework, we clarify relevant interconnections, iterations, sequences, and timing of decision points, stakeholders, and necessary conditions to facilitate an outcome of successful repair at the individual level, and thus intervention strategies for scaling repair within CE. We discuss how a policy mix can address the life cycle of products and the repair system more holistically. We conclude with a future outlook on how temporal dimensions can inform policy strategies and future research.     

Primate terminal complement inhibitor homologues of human CD59

The nucleotide sequence data reported in this paper have been submitted to the GenBank nucleotide sequence database and have been assigned the accession numbers L22862 (African green monkey CD59) and L22863 (baboon CD59)     

Applications of DNA shuffling to pharmaceuticals and vaccines

DNA shuffling is a practical process for directed molecular evolution which uses recombination to dramatically accelerate the rate at which one can evolve genes. Single and multigene traits that require many mutations for improved phenotypes can be evolved rapidly. DNA shuffling technology has been significantly enhanced in the past year, extending its range of applications to small molecule pharmaceuticals, pharmaceutical proteins, gene therapy vehicles and transgenes, vaccines and evolved viruses for vaccines, and laboratory animal models.     

Energy-dispersive X-ray analysis of the extracellular cadmium sulfide crystallites of Klebsiella aerogenes

Klebsiella aerogenes forms electron-dense partieles on the cell surface in response to the presence of cadmium ions in the growth medium. These particles ranged from 20 to 200 nm in size, and quantitative energy dispersive X-ray analysis established that they comprise cadmium and sulfur in a 1:1 ratio. This observation leads to the conclusion that the particles are cadmium sulfide crystallites. A combination of atomic absorption spectroscopy, inductively coupled plasma mass spectrometry, and acid-labile sulfide analysis revealed that the total intracellular and bound extracellular cadmium:sulfur ratio is also 1:1, which suggests that the bulk of the cadmium is fixed as extracellular cadmium sulfide. The tolerance of K. acrogenes to cadmium ions and the formation of the cadmium sulfide crystallites were dependent on the buffer composition of the growth medium. The addition of cadmium ions to phosphate-buffered media resulted in cadmium phosphate precipitates that remove the potentially toxic cadmium ions from the growth medium. Electrondense particles formed on the surfaces of bacteria grown under these conditions were a combination of cadmium sulfide and cadmium phosphates. The specific bacterial growth rate in the exponential phase of batch cultures was not affected by up to 2mM cadmium in Tricine-buffered medium, but formation of cadmium sulfide crystallites was maximal during the stationary phase of batch culture. Cadmium tolerance was much lower (10 to 150 M) in growth media buffered with Tris, Bistris propane, Bes, Tes, or Hepes. These results illustrate the importance of considering medium composition when comparing levels of bacterial cadmium tolerance.Abbreviations EDXA Energy dispersive X-ray analysis - AAS Atomic absorption spectroscopy - TEM Transmission electron microscopy - SEM Scanning electron microscopy - ICP-MS Inductively coupled plasma mass spectrometry - ALSA Acid-labile sulfide analysis     

Influence of 0.5 ppm nitrogen dioxide exposure of mice on macrophage congregation in the lungs

Summary The lungs of 12 mice, half of which were exposed to continuous 0.5 ppm nitrogen dioxide for 3 weeks, were explanted in culture, and the instances of macrophage congregation were quantitated according to numbers of target cells involved, categories of congregation from three to 11 or more, numbers of macrophages participating in each category for the total cultures, and the influence of delaying explantation for 24 and 96 hr. A total of 9042 macrophages and 2140 epithelial and spindle target cells were counted in the outgrowths from 306 explants. The incidence of macrophage congregation (or numbers of target cells) was greater for the cultures from the NO₂-exposed animals, both with respect to total incidences between groups (p→0), and the 0-hr (p<0.001) and 24-hr (p<0.01) culture subgroups. In addition, the values for T₃ to T₆ macrophage congregation were individually and consistently greater for the exposed animal group. Postmortem interval stress at 96 hr appeared to result in large colonies, but they were reduced greatly in number. Also the incidence of macrophage congregation fell by 28% as compared to 0-hr and 24-hr subgroups. Supported by Grants NHLI No. HL 17412 and EPA No. R. 800881.