This article describes ways in which excess nitrogen from fossil fuel combustion may stress the biosphere. Nitrogen emissions can have a direct effect on air quality through both the oxidizing potential of nitrogen oxides and the role these compounds play in the formation of ozone. The complexity of these effects on water quality and on forest nutrition is discussed.

https://harvardforest.fas.harvard.edu/sites/harvardforest.fas.harvard.edu/files/publications/pdfs/Aber_BioScience_1989.pdf

Nitrogen saturation in northern forest ecosystems

The development of nonviral vectors for safe and efficient gene delivery has been gaining considerable attention recently. An ideal nonviral vector must protect the gene against degradation by nuclease in the extracellular matrix, internalize the plasma membrane, escape from the endosomal compartment, unpackage the gene at some point and have no detrimental effects. In comparison to viruses, nonviral vectors are relatively easy to synthesize, less immunogenic, low in cost, and have no limitation in the size of a gene that can be delivered. Significant progress has been made in the basic science and applications of various nonviral gene delivery vectors; however, the majority of nonviral approaches are still inefficient and often toxic. To this end, two nonviral gene delivery systems using either biodegradable poly(D,Llactide-co-glycolide) (PLG) nanoparticles or cell penetrating peptide (CPP) complexes have been designed and studied using A549 human lung epithelial cells. PLG nanoparticles were optimized for gene delivery by varying particle surface chemistry using different coating materials that adsorb to the particle surface during formation. A variety of cationic coating materials were studied and compared to more conventional surfactants used for PLG nanoparticle fabrication. Nanoparticles (~200 nm) efficiently encapsulated plasmids encoding for luciferase (80-90%) and slowly released the same for two weeks. After a delay, moderate levels of gene expression appeared at day 5 for certain positively charged PLG particles and gene expression was maintained for at least two weeks. In contrast, gene expression mediated by polyethyleneimine (PEI) ended at day 5. PLG particles were also significantly less

Nonviral Vectors for Gene Delivery

To produce an immune reaction against a foreign protein usually requires purification of that protein, which is then injected into an animal. The isolation of enough pure protein is time-consuming and sometimes difficult. Here we report that such a response can also be elicited by introducing the gene encoding a protein directly into the skin of mice. This is achieved using a hand-held form of the biolistic system which can propel DNA-coated gold microprojectiles directly into cells in the living animal. Genetic immunization may be time- and labour-saving in producing antibodies and may offer a unique method for vaccination.

Genetic immunization is a simple method for eliciting an immune response.

THE MITOCHONDRIAL GENETIC SYSTEM ....•••••• ••••••••••.• ••••••••••..•......... 291 Organization and Expression . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ... . . . . . . . . . . 291 Genetic Content 296 Regulation of Mitochondrial Gene Expression . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 300

Biogenesis of Mitochondria

▪ Abstract The eutrophication of many ecosystems in recent decades has led to an increased interest in the ecology of nitrogen transformation. Chemolitho-autotrophic ammonia-oxidizing bacteria are responsible for the rate-limiting step of nitrification in a wide variety of environments, making them important in the global cycling of nitrogen. These organisms are unique in their ability to use the conversion of ammonia to nitrite as their sole energy source. Because of the importance of this functional group of bacteria, understanding of their ecology and physiology has become a subject of intense research over recent years. The monophyletic nature of these bacteria in terrestrial environments has facilitated molecular biological approaches in studying their ecology, and progress in this field has been rapid. The ammonia-oxidizing bacteria of the β-subclass Proteobacteria have become somewhat of a model system within molecular microbial ecology, and this chapter reviews recent progress in our knowledge of ...

Ammonia-Oxidizing Bacteria: A Model for Molecular Microbial Ecology

We report here a novel phenomenon, namely that nucleic acids can be delivered into plant cells using high-velocity microprojec-tiles. This research was conducted in the hope of circumventing some of the inherent limitations of existing methods for delivering DNA into plant cells1–6. After being accelerated, small tungsten particles (microprojectiles) pierce cell walls and membranes and enter intact plant cells without killing them. Microprojectiles were used to carry RNA or DNA into epidermal tissue of onion and these molecules were subsequently expressed genetically. This approach can therefore be used to study the transient expression of foreign genes in an intact tissue. It remains to be shown that smaller cell types, as are found in regenerable plant tissues, can be stably transformed by this method. If this proves possible, it would appear to provide a broadly applicable transformation mechanism capable of circumventing the host-range restrictions of Agrobacterium tumefaciens1, and the regeneration problems of protoplast transformation2–5.

High-velocity microprojectiles for delivering nucleic acids into living cells

Biologists commonly wish to introduce a wide range of substances into living cells. Such substances include biological stains, proteins (antibodies or enzymes), and genetic material (either RNA or DNA). The cell membrane and, in the case of plant ceils, the cell wall pose formidable barriers which exclude many macromolecules. The concept of particle bombardment has been put forward as a universal mechanism for transporting substances into any living cell. An acceleration device has been designed and constructed which can accelerate small tungsten particles (1 to 4 urn in diameter) to velocities of about 1,000 to 2,000 ft/sec. We have found that these particles can penetrate cell walls and membranes and enter cells in a nonlethal manner. Thousands of cells can be penetrated simultaneously, in situ, as they occur in tissues. Particle bombardment has been shown to be effective in delivering foreign substances into a variety of plant species, including onion, tobacco, corn, and rice. This new method ...

Delivery of substances into cells and tissues using a particle bombardment process

This report describes a process for delivering foreign genes into maize cells that does not require the removal of cell walls and is capable of delivering DNA into embryogenic and nonembryogenic tissues. Plasmid harboring a chimeric chloramphenicol acetyltransferase (CAT) gene was adsorbed to the surface of microscopic tungsten particles (microprojectiles). These microprojectiles were then accelerated to velocities sufficient for penetrating the cell walls and membranes of maize cells in suspension culture. High levels of CAT activity were consistently observed after bombardment of cell cultures of the cultivar Black Mexican Sweet, which were comparable to CAT levels observed after electroporation of protoplasts. Measurable increases in CAT levels were also observed in two embryogenic cell lines after bombardment. Gene expression was observed only when an intron from the alcohol dehydrogenase 1 gene of maize was ligated between the 35S promoter and the CAT coding region. CAT activity was detected in cell cultures bombarded with microprojectiles with an average diameter of 1.2 μm, but not after bombardment with microprojectiles 0.6 or 2.4 μm in diameter. Bombarding the same sample several times was found to markedly enhance CAT activity. These results demonstrate that the particle bombardment process can be used to deliver foreign DNA into intact cells of maize. Because this process circumvents the difficulties associated with regenerating whole plants from protoplasts, the particle bombardment process may provide significant advantages over existing DNA delivery methods for the production of transgenic maize plants. In addition, the process should be of value for studying transient and stable gene expression within intact cells and tissues.

https://www.pnas.org/content/pnas/85/12/4305.full.pdf

Transfer of foreign genes into intact maize cells with high-velocity microprojectiles

The development of an efficient transformation system is a prerequisite for the molecular analysis of gene expression in plants. In crop plants, this development has been hindered by difficulties encountered both in whole plant regeneration from protoplasts and in the general insusceptibility of monocots to Agrobacterium-mediated transformation. We have circumvented these difficulties by transferring foreign genes directly into the intact cells (with cell walls) of three important crop plants including rice, wheat and soybean by a particle bombardment device. Oryza sativa and Triticum monococcum cells were bombarded with accelerated tungsten particles coated with plasmids containing a β-glucuronidase gene as the reporter. Blue transformed cells were detected in an in situ enzyme assay. The number of blue cells was next used as a convenient criterion to study several factors affecting gene transfer efficiency. After optimal conditions were defined, gene transfer into intact cells of O. sativa, T. monococcum and Glycine max was successfully carried out with chloramphenicol acetyltransferase (CAT) gene as the reporter.

Transient expression of foreign genes in rice, wheat and soybean cells following particle bombardment.

Nitrate was formed from ammonium at pH 3.2 to 6.1 in suspensions of a naturally acid forest soil; the maximum rates of formation occurred at ca. pH 4 to 5. Nitrate was also formed from soil nitrogen in suspensions incubated at 50 degrees C. Autotrophic nitrifying bacteria could not be isolated from this soil. Enrichment cultures produced nitrate in a medium with beta-alanine if much soil was added to the medium, and nitrite but not nitrate was formed in the presence of small amounts of soil. Nitrification by these enrichments was abolished by eucaryotic but not procaryotic inhibitors. A strain of Absidia cylindrospora isolated from this soil was found to produce nitrate and nitrite in a medium with beta-alanine at pH values ranging from 4.0 to 4.8. Nitrate production by A. cylindrospora required the presence of sterile soil. Free and bound hydroxylamine, hydroxamic acids, and primary aliphatic nitro compounds did not accumulate during the conversion of beta-alanine to nitrite by the fungus. The organism also formed nitrite from ammonium in a medium containing acetate. We suggest that nitrification in this soil is a heterotrophic process catalyzed by acid-tolerant fungi and not by autotrophs or heterotrophs in nonacid microsites.

Theodore M. Klein

Papers

High-velocity microprojectiles for delivering nucleic acids into living cells

Delivery of substances into cells and tissues using a particle bombardment process

Transfer of foreign genes into intact maize cells with high-velocity microprojectiles

Transient expression of foreign genes in rice, wheat and soybean cells following particle bombardment.

Heterotrophic Nitrification in an Acid Forest Soil and by an Acid-Tolerant Fungus