The effect of aging on the physical stability of liposome dispersions.

In this study, the effect of aging, in terms of hydrolytic decomposition of the bilayer forming (phospho)lipids, on the physical stability of aqueous liposome dispersion was investigated in partially hydrogenated egg phosphatidylcholine (PHEPC) and egg phosphatidylglycerol (EPG) containing liposomes with or without cholesterol. The physical stability of the liposome dispersions was assessed by measuring the leak-in rate of a non-bilayer interacting hydrophilic marker molecule, calcein and changes in the particle size and its distribution in time. Additionally, permeability of either partially hydrolysed phospholipids or exogenous lyso-phosphatidylcholine(LPC) containing bilayers was calculated. The experiments were performed at 40 degrees C. Liposome dispersions were aged artificially by storing at 60 degrees C. The size of the liposomes and polydispersity index of the dispersions, in general, did not change significantly. The leak-in rate of calcein in externally added LPC containing liposomes was increased relative to the incorporated LPC concentration. The higher the LPC content of the bilayers, the higher the leak-in rate of calcein into liposomes. The leak-in rate of calcein, however, decreased first in partially hydrolysed phospholipids containing liposomes up to around 10% of hydrolysis and, afterwards, it started to increase. The leak-in rate was always lower in partially hydrolysed phospholipids containing liposomes than externally added LPC containing ones. Furthermore, the permeability of cholesterol containing bilayers was also always lower than the bilayers without cholesterol. In conclusion, addition of LPC into liposomal bilayers increases the permeability of bilayer. However, bilayers containing the hydrolysis products of phospholipids, both lyso-phospholipids and free fatty acids, did not show any enhanced permeability up to around 15% hydrolysis. Bilayer permeability is enhanced above 15% hydrolysis.     

Resistance to three thrips species in Capsicum spp. depends on site conditions and geographic regions

Capsicum species are commercially grown for pepper production. This crop suffers severely from thrips damage and the identification of natural sources of thrips resistance is essential for the development of resistant cultivars. It is unclear whether resistance to Frankliniella occidentalis as assessed in a specific environment holds under different conditions. Additionally, other thrips species may respond differently to the plant genotypes. Screening for robust and general resistance to thrips encompasses testing different Capsicum accessions under various conditions and with different thrips species. We screened 11 Capsicum accessions (C. annuum and C. chinense) for resistance to F. occidentalis at three different locations in the Netherlands. Next, the same 11 accessions were screened for resistance to Thrips palmi and Scirtothrips dorsalis at two locations in Asia. This resulted in a unique analysis of thrips resistance in Capsicum at five different locations around the world. Finally, all accessions were also screened for resistance to F. occidentalis in the Netherlands using a leaf disc choice assay, allowing direct comparison of whole plant and leaf disc assays. Resistance to F. occidentalis was only partially consistent among the three sites in the Netherlands. The most susceptible accessions were consistently susceptible, but which accession was the most resistant differed among sites. In Asia, one C. chinense accession was particularly resistant to S. dorsalis and T. palmi, but this was not the most resistant accession to F. occidentalis. Overall, resistance to F. occidentalis correlated with S. dorsalis but not with T. palmi resistance in the C. annuum accessions. Damage inflicted on leaf discs reflected damage on the whole plant level. Our study showed that identifying broad spectrum resistance to thrips in Capsicum may prove to be challenging. Breeding programmes should focus on developing cultivars suitable for growing in defined geographic regions with specific thrips species and abiotic conditions.     

Adenovirus in Rural Côte D`Ivoire: High Diversity and Cross-Species Detection

The Taï region in Western Côte d`Ivoire is characterized by extensive overlap of human and animal habitats. This could influence patterns of adenovirus transmission between humans and domestic animals. Fecal samples from humans and various domestic animals were tested for the presence of adenoviruses by PCR. Phylogenetic and species delineation analyses were performed to further characterize the adenoviruses circulating in the region and to identify potential cross-species transmission events. Among domestic animals, adenovirus shedding was frequent (21.6% of domestic mammals and 41.5% of chickens) and the detected strains were highly diverse, several of them representing novel types. Although no evidence for zoonotic transmission of animal adenovirus was obtained, the present study provides concordant evidence in favor of common cross-species transmission of adenoviruses between different animal species and first indications for adenovirus transmission from humans to animals. These findings underline the thus far underestimated importance of reverse zoonotic transmission of viruses and of the role of domestic animals as pathogen reservoirs, “bridge species,” or intermediate hosts.     

Biochemical characterization and mutational analysis of the mononuclear non-haem Fe2+ site in Dke1, a cupin-type dioxygenase from Acinetobacter johnsonii

beta-diketone-cleaving enzyme Dke1 is a homotetrameric Fe2+-dependent dioxygenase from Acinetobacter johnsonii. The Dke1protomer adopts a single-domain beta-barrel fold characteristic of the cupin superfamily of proteins and features a mononuclear non-haem Fe2+ centre where a triad of histidine residues, His-62, His-64 and His-104, co-ordinate the catalytic metal. To provide structure-function relationships for the peculiar metal site of Dke1 in relation to the more widespread 2-His-1-Glu/Asp binding site for non-haem Fe2+,we replaced each histidine residue individually with glutamate and asparagine and compared binding of Fe2+ and four non-native catalytically inactive metals with purified apo-forms of wild-type and mutant enzymes. Results from anaerobic equilibrium microdialysis (Fe2+) and fluorescence titration (Fe2+, Cu2+, Ni2+, Mn2+ and Zn2+) experiments revealed the presence of two broadly specific metal-binding sites in native Dke1 that bind Fe2+ with a dissociation constant (Kd) of 5 microM (site I) and approximately 0.3 mM (site II). Each mutation, except for the substitution of asparagine for His-104, disrupted binding of Fe2+, but not that of the other bivalent metal ions, at site I,while leaving metal binding at site II largely unaffected. Dke1 mutants harbouring glutamate substitutions were completely inactive and not functionally complemented by external Fe2+.The Fe2+ catalytic centre activity (kcat) of mutants with asparagine substitution of His-62 and His-104 was decreased 140- and 220-fold respectively, compared with the kcat value of 8.5 s(-1) for the wild-type enzyme in the reaction with pentane-2,4-dione.The H64N mutant was not catalytically competent, except in the presence of external Fe2+ (1 mM) which elicited about 1/1000 of wild-type activity. Therefore co-ordination of Fe2+ by Dke1 requires an uncharged metallocentre, and three histidine ligands are needed for the assembly of a fully functional catalytic site. Oxidative inactivation of Dke1 was shown to involve conversion of enzyme-bound Fe2+ into Fe3+, which is then released from the metal centre.     

Complex sex determination in the stinging nettle Urtica dioica

The dioecious species Urtica dioica harbours wide variation in sex ratio of seeds. We conducted a series of crosses to analyse the genetic basis of sex determination in this species. Dutch populations of U. dioica contain low proportions of monoecious individuals beside male and female plants. Self-pollination of monoecious plants always yielded female, male and monoecious plants, generally in a ratio of one female to three male/monoecious individuals. This motivated us to write down a simple model in which gender is determined by one major sex-determination locus with four alleles. In the model males and monoecious plants have distinct genotypes but are both heterozygous at the sex-determination locus. We first made crosses among progeny obtained after self-pollination of monoecious plants. These crosses showed that the monoecious trait generally showed Mendelian inheritance and was passed on to the next generation via both pollen and seeds. Further crosses between monoecious plants and plants from dioecious system indicated that alleles from the dioecious system are often dominant. However, many exceptions to our genetic model are observed which suggest that dominance is incomplete and/or that more genes are involved in sex determination. We discuss to what extent sex determination genes explain the strongly biased seed sex ratios and argue that additional genes, for instance genes for female choice, must also be involved.     

Amyloid precursor protein expression is induced by tumor necrosis factor α in 3T3‐L1 adipocytes

Amyloid precursor protein (APP) has been characterized as an adipocyte‐secreted protein that might contribute to obesity‐related insulin resistance, inflammation, and dementia. In the current study, regulation of APP by the proinflammatory and insulin resistance‐inducing cytokine tumor necrosis factor (TNF) α was determined in 3T3‐L1 adipocytes. Interestingly, APP protein synthesis and mRNA expression were significantly increased by TNFα in a time‐dependent manner with maximal induction observed after 24 h of treatment. Furthermore, TNFα induced APP mRNA expression dose‐dependently with maximal 6.4‐fold upregulation seen at 100 ng/ml effector. Moreover, inhibitor experiments suggested that TNFα‐induced APP expression was mediated by nuclear factor κ B. Taken together, we show for the first time a potent upregulation of APP by TNFα suggesting a potential role of this adipocyte‐secreted protein in TNFα‐induced insulin resistance and inflammatory disease. J. Cell. Biochem. 108: 1418–1422, 2009. © 2009 Wiley‐Liss, Inc.     

Association mapping reveals gene action and interactions in the determination of flowering time in barley

The interaction between members of a gene network has an important impact on the variation of quantitative traits, and can influence the outcome of phenotype/genotype association studies. Three genes (Ppd-H1, HvCO1, HvFT1) known to play an essential role in the regulation of flowering time under long days in barley were subjected to an analysis of nucleotide diversity in a collection of 220 spring barley accessions. The coding region of Ppd-H1 was highly diverse, while both HvCO1 and HvFT1 showed a rather limited level of diversity. Within all three genes, the extent of linkage disequilibrium was variable, but on average only moderate. Ppd-H1 is strongly associated with flowering time across four environments, showing a difference of five to ten days between the most extreme haplotypes. The association between flowering time and the variation at HvFT1 and HvCO1 was strongly dependent on the haplotype present at Ppd-H1. The interaction between HvCO1 and Ppd-H1 was statistically significant, but this association disappeared when the analysis was corrected for the geographical origin of the accessions. No association existed between flowering time and allelic variation at HvFT1. In contrast to Ppd-H1, functional variation at both HvCO1 and HvFT1 is limited in cultivated barley.     

Why do shrews twitter? Communication or simple echo-based orientation

Shrews are very vocal animals. We tested behaviourally whether the high-pitched laryngeal ‘twittering’ calls of as-yet unclear function serve for communication or echo-based orientation. We used a representative species from each of the two largest phylogenetic groups of shrews. In both species, experimental manipulation of substrate density, but not of the likelihood of conspecific presence, affected the shrews'' call rate when exploring an unknown environment. This adaptation of call rate to the degree of habitat clutter parallels bat echolocation and suggests that shrews may use the echoes and reverberations of their calls for identifying routes through their habitat or for probing habitat type. To assess the acoustic feasibility of shrew echo orientation, we ensonified shrew habitats in the field with an ‘artificial shrew’ (small speaker mounted close to a sensitive microphone). The data showed that shrew-like calls can indeed yield echo scenes useful for habitat assessment at close range, but beyond the range of the shrews'' vibrissae.     

A Systematic Proteomic Study of Irradiated DNA Repair Deficient Nbn-Mice

Background

The NBN gene codes for the protein nibrin, which is involved in the detection and repair of DNA double strand breaks (DSBs). The NBN gene is essential in mammals.

Methodology/Principal Findings

We have used a conditional null mutant mouse model in a proteomics approach to identify proteins with modified expression levels after 4 Gy ionizing irradiation in the absence of nibrin in vivo. Altogether, amongst ∼8,000 resolved proteins, 209 were differentially expressed in homozygous null mutant mice in comparison to control animals. One group of proteins significantly altered in null mutant mice were those involved in oxidative stress and cellular redox homeostasis (p<0.0001). In substantiation of this finding, analysis of Nbn null mutant fibroblasts indicated an increased production of reactive oxygen species following induction of DSBs.

Conclusions/Significance

In humans, biallelic hypomorphic mutations in NBN lead to Nijmegen breakage syndrome (NBS), an autosomal recessive genetic disease characterised by extreme radiosensitivity coupled with growth retardation, immunoinsufficiency and a very high risk of malignancy. This particularly high cancer risk in NBS may be attributable to the compound effect of a DSB repair defect and oxidative stress.