We have characterized the relationships between the design of cationic liposomes as a gene transfer vehicle, their resulting biodistribution and processing in animals, and the level and sites of gene expression they produce. By redesigning conventional cationic liposomes, incorporating cholesterol (chol) as the neutral lipid and preparing them as multilamellar vesicles (MLV), we increased the efficiency of cationic liposome:DNA complex (CLDC)-mediated gene delivery. Expression of the luciferase gene increased up to 1,740-fold and of the human granulocyte-colony stimulating factor (hG-CSF) gene up to 569-fold due to prolonged circulation time of injected CLDC, and increased uptake and retention in tissues. The level of gene expression per microgram of DNA taken up per tissue was 1,000-fold higher in lung than in liver, indicating that in addition to issues of delivery and retention of injected DNA, tissue-specific host factors also play a central role in determining the efficiency of expression. Vascular endothelial cells, monocytes, and macrophages are the cell types most commonly transfected by intravenous injection of CLDC.

We have developed three catioinic amphiphiles based on the structure 1-[2-(acyloxy)ethyl]-2-alkyl(alkenyl)-3-(2-hydroxyethyl)imidazolinium chloride. Although these three compounds differ only in the structure of the hydrophobic acyl chains, they differ greatly in their ability to mediate in vivo and in vitro gene delivery. Moreover, in vitro efficiency is not predictive of in vivo efficiency. The myristoyl form is the most effective compound in vitro, and the oleoyl form is the most effective compound in vivo. The compounds readily form suspensions in aqueous media, both in the pure form and as mixtures with either cholesterol or dioleoylphosphatidylethanolamine. These suspensions can be sonicated to produce smaller particles. Particle size, electron microscopy, and the ability to capture glucose suggest that these lipids form liposomes on suspension in aqueous media. When mixed with plasmid DNA, the lipid particles appear to fuse and form larger particles. Fusion is maximal at the critical DNA:lipid ratio where extensive aggregation and precipitation are observed. Therefore, these compounds behave similarly to other cationic liposome-forming lipids upon interaction with DNA.

Rnf proteins are proposed to form membrane-protein complexes involved in the reduction of target proteins such as the transcriptional regulator SoxR or the dinitrogenase reductase component of nitrogenase. In this work, we investigate the role of rnf genes in the nitrogen-fixing bacterium Azotobacter vinelandii. We show that A. vinelandii has two clusters of rnf-like genes: rnf1, whose expression is nif-regulated, and rnf2, which is expressed independently of the nitrogen source in the medium. Deletion of each of these gene clusters produces a time delay in nitrogen-fixing capacity and, consequently, in diazotrophic growth. ⌬rnf mutations cause two distinguishable effects on the nitrogenase system: (i), slower nifHDK gene expression and (ii), impairment of nitrogenase function. In these mutants, dinitrogenase reductase activity is lowered, whereas dinitrogenase activity remains essentially unaltered. Further analysis indicates that ⌬rnf mutants accumulate an inactive and iron-deficient form of NifH because they have lower rates of incorporation of [4Fe-4S] into NifH. ⌬rnf mutations also cause a noticeable decrease in aconitase activity; however, they do not produce general oxidative stress or modification of Fe metabolism in A. vinelandii. Our results suggest the existence of a redox regulatory mechanism in A. vinelandii that controls the rate of expression and maturation of nitrogenase by the activity of the Rnf protein complexes. rnf1 plays a major and more specific role in this scheme, but the additive effects of mutations in rnf1 and rnf2 indicate the existence of functional complementation between the two homologous systems.iron-sulfur cluster ͉ NifH ͉ rnf T he conversion of N 2 gas to ammonia is called nitrogen fixation. The ability to fix nitrogen, which is confined to prokaryotes, constitutes a crucial part of the nitrogen cycle in the biosphere that is necessary to sustain life on Earth. The great majority of biological nitrogen fixation is catalyzed by the molybdenum-containing nitrogenase, which is composed of two proteins that can be separately purified: dinitrogenase and dinitrogenase reductase. Dinitrogenase, also referred to as the MoFe protein or component I, is a 220-to 240-kDa tetramer of the nifD and nifK (nif genes encode proteins involved in nitrogen fixation) gene products that contains two pairs of two complex metalloclusters known as the P cluster and the iron molybdenum cofactor (FeMo-co). Dinitrogenase reductase, also referred to as the Fe protein or component II, is a 60-kDa dimer of the product of the nifH gene, which contains a single cluster at the subunit interface and two Mg-ATP binding sites, one at each subunit. The Fe protein is the obligate electron donor to the MoFe protein; electrons are transferred from the [4Fe-4S] cluster of the Fe protein to the P cluster of the MoFe protein and, in turn, to FeMo-co, the site for substrate reduction (1-3).Much effort has been applied to the study of nitrogenase metallocluster assembly because it is not only relevant to biological nitrog...

Background: The optimal management strategy for multiple sclerosis (MS), and many other chronic diseases, likely involves health behavior modification. Multimodal behavioral interventions may be most effective, but little is known about long-term adherence in people with MS. Methods: This qualitative study assessed barriers and enablers to long-term adherence by people with MS who self-selected for a 5-day health behavior intervention 3-5 years prior. Thirteen women and five men participated in semi-structured phone interviews, which were transcribed and thematically analyzed. Results: The experience was described as useful for information gathering, decision making, and practical strategies regarding health behaviors. The majority still followed supplementation and dietary recommendations most of the time, although consuming non-recommended food while eating out was common. Support at home, ability and enjoyment in food preparation, and ability to resist unhealthy foods were both barriers and enablers. Adherence to "time-consuming" exercise and meditation recommendations were less common and episodic. Many reported competing interests on time from work and family; and barriers including injuries and symptoms, weather, financial or geographical barriers, and lack of personcentred support and motivation. Increased fitness and mobility, weight loss, and a sense of accomplishment and control were advantages and motivators. Practical and attitudinal strategies employed included planning, tailoring activities to ability and preference, and self-monitoring. Conclusion: While most people attempted to engage with all components of the intervention initially, only some still engaged with all components, and none to the recommended levels. These data can inform future quantitative studies and health behavior interventions. ä IMPLICATIONS FOR REHABILITATION A multimodal group lifestyle intervention may be useful to assist people with multiple sclerosis in information gathering, decision making, attitudinal changes, and practical strategies regarding health behaviors; as well as providing a sense of hope for the future and control over wellbeing. While participants are unlikely to engage with all components of a multimodal intervention to the recommended level, they are likely to make improvements to one or more health behaviors. Experiential learning, including going through recipes and ingredient lists, and practicing meditation and physical exercises, is helpful to instigate behavior change. The initiation and maintenance of behavior change was assisted by support from family, friends, and health practitioners; and practical strategies employed by participants, including planning, self-monitoring, and tailoring activities to ability and preference ARTICLE HISTORY