Interaction of polyamine gene vectors with RNA leads to the dissociation of plasmid DNA-carrier complexes

BACKGROUND: Plasmid DNA (pDNA) dissociation from polyamine gene vectors after cellular uptake has not been well characterized. A more detailed understanding of this process could lead to more efficient gene transfer agents. Since RNA is present in the cytoplasm at high concentrations and due to its structural similarity to DNA, we were interested in its conceivable interaction with polyamine gene vectors. METHODS: In a first set of experiments gene vectors were incubated in cell lysate and pDNA release was investigated by Southern blot analysis with or without RNase A pretreatment and by confocal laser scanning microscopy. Further, interaction of polyamine gene vectors with RNA was investigated by fluorescence quenching assay. These methods were complemented by a functionality assay using isolated nuclei. RESULTS: The incubation of gene vectors with cell lysate resulted in the dissociation of pDNA from the complexes. This effect was abolished when the cell lysate was pretreated with RNase A. The addition of RNA in the absence of cell lysate led also to a dissociation of pDNA. This process commenced instantaneously after the addition of RNA as analyzed by fluorescence quenching. When gene vectors were incubated in cell lysate containing isolated nuclei, the dissociation of pDNA from the polyamine gene vectors occurred preferentially extranuclearally as confirmed by confocal laser scanning microscopy. These results were further corroborated in a functional assay. CONCLUSIONS: These data suggest that RNA induces pDNA dissociation from the polyamine gene vectors. Furthermore, this process apparently occurs in the cytoplasm before the gene vectors enter the nucleus.     

Shear field mapping in actin networks by using magnetic tweezers

An improved magnetic bead microrheometer based on phase contrast microscopy allowing high resolution measurements of local deformations within macromolecular networks is applied to study local viscoelastic properties of cross-linked actin networks. By embedding non-magnetic colloidal beads as probes into the networks, the spatial variation of the strain field within cross-linked actin networks can be mapped. Moreover, the Poisson ratio and shear modulus can be measured locally.     

Lipophilic constituents from aerial and root parts of Mercurialis perennis L.

Introduction – Dog's mercury (Mercurialis perennis L.) is a perennial herb used in remedies for medicinal purposes. The plant is supposed to contain potentially active substances but its constituents have only been rarely studied. Objective – Detailed studies on the phytochemical composition are of great interest to broaden the knowledge on the chemotaxonomy and pharmacognosy of M. perennis. Methodology – Chloroform and hexane extracts from roots and aerial parts were investigated using GC/MS and LC/MS. Results – The whole plant exhihited a broad spectrum of structurally diverse constituents, mainly alkaloids, terpenes, sterols and simple aromatic compounds. Closer inspection of the piperidine alkaloid hermidin revealed its inherent instability towards air oxygen. To obtain quantitative data on these alkaloids the synthesis of the more stable reference compound 4‐methoxy‐1‐methylpyridine‐2,6(1H,3H)‐dione (MMPD) was required. In this study, MMPD was detected for the first time as a genuine compound in Mercurialis. Hermidine quinone and hermidin dimers originating from hermidin via a free anionic radical reaction were also confirmed by GC/MS. Moreover, volatile compounds such as benzylalcohol, 2‐phenylethanol, 4‐methoxy‐ and 3,4‐dimethoxyphenol, (?)‐cis‐ and (+)‐trans‐myrtanol, (?)‐cis‐myrtanal as well as squalene were predominantely present in Mercurialis roots. In contrast, aerial parts mainly contained phytol derivatives, sterols and tocopherols. By changing solvent polarity, lipid and wax‐containing fractions were obtained. LC/MS‐studies on hexane extracts showed the presence of several mixed triglycerides constituted by linolenic, linoleic, oleic, stearic and palmitic acids, as well as lutein, carotenes and pheophytins. Conclusions – The phytochemical data presented complement our knowledge on the rarely studied plant M. perennis and may broaden its use in future phytotherapy. Copyright © 2009 John Wiley & Sons, Ltd.     

SPORE RELEASE IN ACROCHAETIUM SP. (RHODOPHYTA) IS BACTERIALLY CONTROLLED1

The facultative red algal epiphyte Acrochaetium sp. liberated spores preferentially and recruited more successfully in laboratory cultures when its host Gracilaria chilensis C. J. Bird, McLachlan et E. C. Oliveira was present. The same effect was also induced by cell‐free medium from G. chilensis, suggesting it contained a molecular signal. Antibiotics prevented spore release in Acrochaetium sp., even when G. chilensis was present, suggesting a prokaryotic origin of the signal. Simultaneous application of N‐butyl‐homoserine‐lactone (BHL) restored the spore‐release capacity, which demonstrated that spore release was not directly inhibited by the antibiotics and indicated that bacterially generated N‐acyl‐homoserine‐lactones (AHLs) regulate spore release. An involvement of AHL was further indicated by the fact that two different halofuranone inhibitors of AHL receptors also inhibited spore release when they were applied at relatively low concentrations. Of seven different AHLs tested, only BHL induced the effect. However, BHL was only active at relatively high concentrations (100 μM), and it was not detected in spore‐release‐inducing medium of G. chilensis. Another water‐soluble AHL or an AHL structure analog is therefore probably the active compound in G. chilensis cultures. The data presented demonstrate that life cycle completion in Acrochaetium sp. strongly depends on bacteria, which are not always present in sufficient numbers on the alga itself. Exogenous bacteria that are associated with G. chilensis or with other potential substrates may therefore trigger timely spore liberation in Acrochaetium sp., provided that the necessary concentration of AHL is reached. This first finding of AHL perception in a red alga confirms that AHL signalling is more widespread among eukaryotes than was thought until recently. However, spore release of a second red alga, Sahlingia subintegra (Rosenv.) Kornmann, was unaffected by AHL, and the reaction observed is therefore not universal.     

Land use affects rodent communities in Kalahari savannah rangelands

Shrub encroachment caused by overgrazing has led to dramatic changes of savannah landscapes and is considered one of the most threatening forms of rangeland degradation leading to habitat fragmentation. Although changes to plant assemblages are becoming better known, however, our understanding of how shrub encroachment affects rodent communities is low. In this study, we investigated relative abundance of five rodent species in sixteen southern Kalahari rangelands where shrub cover ranged from low (<5%) to high (>25%). Rodent abundance was determined on three trapping grids (40 × 100 m) for each site. Our results show that increasing shrub cover affected rodent species differently. The relative abundance of hairy‐footed gerbil, short‐tailed gerbil and bushveld gerbil declined with increasing shrub cover, whereas highveld gerbil and striped mouse exhibited hump‐shaped relationships with shrub cover. Overall, species richness decreased with increasing shrub cover and a negative impact of high shrub cover above 15% on rodent abundance was congruent for all species. We conclude that our results support the hypothesis that long‐term heavy grazing that results in area wide shrub encroachment, threatens the diversity of arid environments.     

Structure of binuclear Rh(II) carboxylato complexes with 2,2-bipyridine and an unprecedented rhodium wire with Rh4 core

Structures of rhodium(II) binuclear complexes [Rh₂(OOCCH₃)₂(bpy)₂(H₂O){(CH₃)₂CHOH}][B(C₆H₅)₄]₂ · H₂O (1), [Rh₂Cl₂(OOCCH₃)₂(bpy)₂] · 2H₂O (2), [Rh₂Br₂(OOCCH₃)₂(bpy)₂] · 3H₂O (3), and [Rh₂I₂(OOCCH₃)₂(bpy)₂] (4), as well as an unprecedented wire with infinite Rh-Rh chain, {[Rh₄(μ-OOCH)₄(bpy)₄](BF₄)}_n · 0.5nC₄H₈O₂ (5), have been determined and discussed. Mass spectra of complexes [Rh₂(OOCMe)₂(bpy)₂(H₂O)₂](MeCOO)₂ and [Rh₂(OOCMe)₂(phen)₂(H₂O)₂](MeCOO)₂ have showed stability of polynuclear cations with rhodium in oxidation states in the range +1.25 to +1.75.     

Cytotoxicity of RNases is increased by cationization and counteracted by K(Ca) channels

K(Ca) channels are involved in control of cell proliferation and differentiation. Here we have revealed their role in overcoming the RNase-induced cytotoxicity. Toxic effects of Streptomyces aureofaciens RNases Sa, Sa2, Sa3, and of RNase Sa charge reversal mutants on the human embryonic kidney cell lines differing only by the presence of K(Ca) channels were characterized. In contrast to other RNases, a basic variant of RNase Sa and RNase Sa3 exhibit significant cytotoxic activity of the same order of magnitude as onconase. Our data indicate the absence of a correlation between catalytic activity and stability of RNases and cytotoxicity. On the other hand, cationization enhances toxic effect of an RNase indicating the major role of a positive charge. Essentially lower sensitivity to cytotoxic microbial RNases of cells expressing K(Ca) channels was found. These results suggest that cells without the K(Ca) channel activity cannot counteract toxic effect of RNases.     

Complex III releases superoxide to both sides of the inner mitochondrial membrane

Mechanisms of mitochondrial superoxide formation remain poorly understood despite considerable medical interest in oxidative stress. Superoxide is produced from both Complexes I and III of the electron transport chain, and once in its anionic form it is too strongly charged to readily cross the inner mitochondrial membrane. Thus, superoxide production exhibits a distinct membrane sidedness or "topology." In the present work, using measurements of hydrogen peroxide (Amplex red) as well as superoxide (modified Cypridina luciferin analog and aconitase), we demonstrate that Complex I-dependent superoxide is exclusively released into the matrix and that no detectable levels escape from intact mitochondria. This finding fits well with the proposed site of electron leak at Complex I, namely the iron-sulfur clusters of the (matrix-protruding) hydrophilic arm. Our data on Complex III show direct extramitochondrial release of superoxide, but measurements of hydrogen peroxide production revealed that this could only account for approximately 50% of the total electron leak even in mitochondria lacking CuZn-superoxide dismutase. We posit that the remaining approximately 50% of the electron leak must be due to superoxide released to the matrix. Measurements of (mitochondrial matrix) aconitase inhibition, performed in the presence of exogenous superoxide dismutase and catalase, confirmed this hypothesis. Our data indicate that Complex III can release superoxide to both sides of the inner mitochondrial membrane. The locus of superoxide production in Complex III, the ubiquinol oxidation site, is situated immediately next to the intermembrane space. This explains extramitochondrial release of superoxide but raises the question of how superoxide could reach the matrix. We discuss two models explaining this result.