Mechanism of naked DNA clearance after intravenous injection

BACKGROUND: Injection of naked DNA has been viewed as a safer alternative to current gene delivery systems; however, the rate of clearance from the circulation has been a constant barrier in developing these methods. Naked DNA after intravenous (i.v.) injection will be taken up by the liver and depredated by serum nucleases. MATERIALS AND METHODS: Our study examines the mechanisms involved in clearance of naked DNA by each compartment, the blood and the liver, in an in vivo mouse model. Confocal microscopy and transmission electron microscopy were employed to identify the type of cells taking up DNA and the barrier to DNA access to hepatocytes, respectively. RESULTS: Our data showed the liver could take up over 50% of 5 microg perfused pDNA, with a maximum 25 microg of pDNA during a single pass, and a slower clearance rate compared to that of liver uptake. It was demonstrated that naked DNA is primarily taken up by the liver endothelial cells and this endothelial barrier to transfection could be overcome by manually massaging the liver, which enlarges the fenestrae. CONCLUSIONS: This study clarifies the mechanism by which naked DNA is eliminated from the circulation after i.v. injection, focusing on the role of both the liver and blood compartments in vivo (i.e. mouse). With this knowledge, we can more clearly understand the mechanism of naked DNA clearance and develop more efficient strategies for DNA transfer in vivo.     

裸燕麦EMS突变体库筛选与分析

燕麦是重要的粮饲兼用作物,构建燕麦EMS突变体库对燕麦功能基因组学研究和遗传改良有重要意义。本试验利用化学诱变剂甲基磺酸乙酯(EMS,ethyl methane sulfonate)处理燕麦品种花早2号,获得了4083株M1材料;对其中2000个单株种植了M2株行,进行全生育期调查,鉴定其表型变化;对2份黄化苗突变材料种植了M3家系,进行相关突变性状的稳定性验证。结果表明,燕麦经EMS处理后代变异巨大,在M2发现表型突变材料196份,变异率为9.8%,变异类型非常丰富,包括幼苗习性、叶片性状、分蘖、株高、穗部形态及成熟期等突变株系。M3证实突变的黄化苗特性可以稳定遗传。本研究建立了燕麦EMS诱变体系,获得的燕麦变异类型丰富,为燕麦功能基因组学研究和燕麦遗传改良奠定了材料基础。     

Metabolic clues to salubrious longevity in the brain of the longest‐lived rodent: the naked mole‐rat

Naked mole‐rats (NMRs) are the oldest‐living rodent species. Living underground in a thermally stable ecological niche, NMRs have evolved certain exceptional traits, resulting in sustained health spans, negligible cognitive decline, and a pronounced resistance to age‐related disease. Uncovering insights into mechanisms underlying these extraordinary traits involved in successful aging may conceivably provide crucial clues to extend the human life span and health span. One of the most fundamental processes inside the cell is the production of ATP, which is an essential fuel in driving all other energy‐requiring cellular activities. Not surprisingly, a prominent hallmark in age‐related diseases, such as neurodegeneration and cancer, is the impairment and dysregulation of metabolic pathways. Using a two‐dimensional polyacrylamide gel electrophoresis proteomics approach, alterations in expression and phosphorylation levels of metabolic proteins in the brains of NMRs, aged 2–24 years, were evaluated in an age‐dependent manner. We identified 13 proteins with altered levels and/or phosphorylation states that play key roles in various metabolic pathways including glycolysis, β‐oxidation, the malate‐aspartate shuttle, the Tricarboxylic Acid Cycle (TCA) cycle, the electron transport chain, NADPH production, as well as the production of glutamate. New insights into potential pathways involved in metabolic aspects of successful aging have been obtained by the identification of key proteins through which the NMR brain responds and adapts to the aging process and how the NMR brain adapted to resist age‐related degeneration.

     

Evolution of nakedness in Homo sapiens

Homo sapiens L. is the only existing primate species lacking in functionally effective thermally insulating fur. As all other primates have considerable hair covering, it has always been accepted that our ancestors must once have had a respectable amount of body hair. Unfortunately, fossils cannot help us when it comes to differences in skin and hair. Recent DNA analysis, however, has given us some idea of when and where the great denudation took place. A number of hypotheses have been proposed to account for this feature, but none of these has gained general acceptance. In this paper, I present these hypotheses in the light of current empirical evidence and discussion.     

Population dynamics of the bird cherry‐oat aphid,Rhopalosiphum padi (L.), during the autumn and winter: a modelling approach

1 A simulation model was developed to investigate the inter‐relationship of factors influencing the population dynamics of the bird cherry‐oat aphid (Rhopalosiphum padi (L.)) in barley crops during the autumn and winter. 2 The model incorporated algorithms describing alate immigration, development and survival of adults and nymphs, fecundity and morph determination in newly born nymphs. 3 The model was validated against pest outbreaks in barley fields in south‐east England. 4 It simulated accurately the size of the outbreaks with predictions of peak aphid populations within 20% of the observed in all but one case. Similarly, all but one of the year‐sowing date combination predictions of timing of peak abundance fell within 14 days of the observed. 5 A sensitivity analysis of the model highlighted the relative importance of various population processes in determining simulated aphid population dynamics; decreasing mortality rates of apterous nymphs by as little as 5% over the autumn and winter increased peak densities by as much as 60‐fold, whereas increasing daily temperatures by only 1 °C more than doubled peak aphid abundance. 6 The model identified our understanding of the mechanisms of aphid mortality as a limiting factor in the accurate prediction of R. padi outbreaks in the field.     

Sustained high levels of neuregulin-1 in the longest-lived rodents; a key determinant of rodent longevity

Naked mole-rats (Heterocephalus glaber), the longest-lived rodents, live 7-10 times longer than similarly sized mice and exhibit normal activities for approximately 75% of their lives. Little is known about the mechanisms that allow them to delay the aging process and live so long. Neuregulin-1 (NRG-1) signaling is critical for normal brain function during both development and adulthood. We hypothesized that long-lived species will maintain higher levels of NRG-1 and that this contributes to their sustained brain function and concomitant maintenance of normal activity. We monitored the levels of NRG-1 and its receptor ErbB4 in H. glaber at different ages ranging from 1 day to 26 years and found that levels of NRG-1 and ErbB4 were sustained throughout development and adulthood. In addition, we compared seven rodent species with widely divergent (4-32 year) maximum lifespan potential (MLSP) and found that at a physiologically equivalent age, the longer-lived rodents had higher levels of NRG-1 and ErbB4. Moreover, phylogenetic independent contrast analyses revealed that this significant strong correlation between MLSP and NRG-1 levels was independent of phylogeny. These results suggest that NRG-1 is an important factor contributing to divergent species MLSP through its role in maintaining neuronal integrity.     

FbpA — A bacterial transferrin with more to offer

Background

Gram negative bacteria require iron for growth and virulence. It has been shown that certain pathogenic bacteria such as Neisseria gonorrhoeae possess a periplasmic protein called ferric binding protein (FbpA), which is a node in the transport of iron from the cell exterior to the cytosol.

Scope of review

The relevant literature is reviewed which establishes the molecular mechanism of FbpA mediated iron transport across the periplasm to the inner membrane.

Major conclusions

Here we establish that FbpA may be considered a bacterial transferrin on structural and functional grounds. Data are presented which suggest a continuum whereby FbpA may be considered as a naked iron carrier, as well as a Fe–chelate carrier, and finally a member of the larger family of periplasmic binding proteins.

General significance

An investigation of the molecular mechanisms of action of FbpA as a member of the transferrin super family enhances our understanding of bacterial mechanisms for acquisition of the essential nutrient iron, as well as the modes of action of human transferrin, and may provide approaches to the control of pathogenic diseases. This article is part of a Special Issue entitled Transferrins: Molecular mechanisms of iron transport and disorders.     

Fiber-rich diets alter rat intestinal leukocytes metabolism

This study addressed the following question: What is the effect of fermentable and nonfermentable fiber-rich diets on intestinal immune cells' function and metabolism? For this purpose, weaning rats received, for 8 weeks, two types of fiber-enriched (30%) diets with different fermentable/nonfermentable fiber ratios, that is, oat bran (0.3) and wheat bran (0.14). The results of these two experimental groups were compared with those of the low-fiber control group having a 0.22 fermentable/nonfermentable fiber ratio. The total number and proportion of leukocytes in plasma, total number of cells in the lymphoid organs, lymphocyte proliferative activity and capacity of phagocytosis, hydrogen peroxide production, and adherence of macrophages were investigated. The activities of key enzymes of glycolysis and glutaminolysis, and of the Krebs cycle of lymphocytes from the mesenteric lymph nodes and macrophages from the intraperitoneal cavity were determined. The metabolic response of lymphocytes and macrophages from rats fed the three diets to Bacillus Calmette-Guérin-stimulus was also investigated. The number of lymphocytes in the mesenteric lymph nodes was lower in both fiber-rich diets than in the control but did not have any difference in the remaining lymphoid organs. Wheat bran caused a significant reduction in the phagocytosis capacity and adherence index of macrophages, whereas oat bran did not have a significant effect. The response of glucose and glutamine metabolism to Bacillus Calmette-Guérin-stimulus was not altered by the diets in lymphocytes, whereas in macrophages, the increase in glutaminase and hexokinase activities was abolished.     

Complete sequence analysis of transgene loci from plants transformed via microprojectile bombardment

A substantial literature exists characterizing transgene locus structure from plants transformed via Agrobacterium and direct DNA delivery. However, there is little comprehensive sequence analysis of transgene loci available, especially from plants transformed by direct delivery methods. The goal of this study was to completely sequence transgene loci from two oat lines transformed via microprojectile bombardment that were shown to have simple transgene loci by Southern analysis. In line 3830, transformed with a single plasmid, one major and one of two minor loci were completely sequenced. Both loci exhibited rearranged delivered DNA and flanking genomic sequences. The minor locus contained only 296 bp of two non-contiguous fragments of the delivered DNA flanked by genomic (filler) DNA that did not originate from the integration target site. Predicted recognition sites for topoisomerase II and a MAR region were observed in the transgene integration target site for this non-functional minor locus. Line 11929, co-transformed with two different plasmids, had a single relatively simple transgene locus composed of truncated and rearranged sequences from both delivered DNAs. The transgene loci in both lines exhibited multiple transgene and genomic DNA rearrangements and regions of scrambling characteristic of complex transgene loci. The similar characteristics of recombined fragments and junctions in both transgenic oat lines implicate similar mechanisms of transgene integration and rearrangement regardless of the number of co-transformed plasmids and the level of transgene locus complexity.