Gene delivery with low molecular weight linear polyethylenimines

BACKGROUND: Linear polyethylenimine (LPEI) with a molecular weight (MW) of 22 kDa has been described as having a superior ability to induce gene transfer compared to its branched form. However, the transfection efficiency of the polymer cannot be enhanced beyond a certain limit due to cytotoxicity. We explored the potential of utilizing LPEIs with MWs ranging from 1.0 to 9.5 kDa to overcome this limitation. METHODS: Polyplexes of plasmid DNA encoding for the enhanced green fluorescent protein (EGFP) and various LPEIs were compared concerning their transfection efficiency and cytotoxicity in CHO-K1 and HeLa cells by flow cytometry. The involvement of endolysosomes in LPEI-mediated gene transfer was investigated by applying the proton pump inhibitor bafilomycin A1 and the lysosomotropic agent sucrose. Confocal laser scanning microscopy was applied to assess the size and shape of polyplexes under cell culture conditions, to detect their endolysosomal localization and to observe their translocation to the nucleus. RESULTS: The transfection efficiency could be altered by varying the MW and the amount of the polymer available for polyplex formation. The highest transfection efficiency (about 44%), i.e. the fraction of EGFP-positive cells, was obtained with LPEI 5.6 kDa, while the cytotoxicity remained low. The colocalization of polyplexes and endolysosomes was observed, and it appeared that the larger polyplexes escaped from the acidic organelles particularly quickly. For LPEI 5.0 and 9.0 kDa, the number of cells and nuclei that had taken up DNA after 6 hours was similar, as determined by flow cytometry. CONCLUSIONS: Our study suggests that LPEIs with low MWs are promising candidates for non-viral gene delivery, because they are more efficient and substantially less toxic than their higher MW counterparts.     

Genetic diversity and differentiation of central European freshwater pearl mussel (Margaritifera margaritifera L.) populations: implications for conservation and management

Despite the fact that mollusc species play an important role in many aquatic ecosystems, little is known about their biodiversity and conservation genetics. Freshwater pearl mussel (Margaritifera margaritifera L.) populations are seriously declining all over Europe and a variety of conservation programs are being established to support the remaining endangered central European populations. In order to provide guidelines for conservation strategies and management programs, we investigated the genetic structure of 24 freshwater pearl mussel populations originating from five major central European drainages including Elbe, Danube, Rhine, Maas and Weser, representing the last and most important populations in this area. We present a nondestructive sampling method of haemolymph for DNA analyses, which is applicable for endangered bivalves. The analyses of nine microsatellite loci with different levels of polymorphism revealed a high degree of fragmented population structure and very different levels of genetic diversity within populations. These patterns can be explained by historical and demographic effects and have been enforced by anthropogenic activities. Even within drainages, distinct conservation units were detected, as revealed from high F(ST) values, private alleles and genetic distance measures. Populations sampled close to contact zones between main drainage systems showed lowest levels of correct assignment to present-day drainage systems. Populations with high priority for conservation should not only be selected by means of census population size and geographical distance to other populations. Instead, detailed genetic analyses are mandatory for revealing differentiation and diversity parameters, which should be combined with ecological criteria for sustainable conservation and recovery programs.     

Reproductive conflict in social insects: male production by workers in a slave-making ant

In insect societies, workers cooperate but may also pursue their individual interests, such as laying viable male eggs. The case of obligatory slave-making ants is of particular interest because workers do not engage in maintenance activities and foraging. Therefore, worker egg laying is expected to be less detrimental for colony efficiency than in related, nonparasitic species. Furthermore, as slave-making workers usually do not perform brood care and thus might have little power in manipulating sex allocation, they might be more strongly selected to increase their direct fitness by producing their own sons than workers in nonparasitic species. In this study we investigated worker reproduction in four natural colonies of the slave-making ant Polyergus rufescens, using highly variable microsatellite markers. Our results show that workers produce up to 100% of the males. This study thus presents the first direct evidence of an almost complete takeover of male reproduction by workers in ants.     

Numb and alpha-Adaptin regulate Sanpodo endocytosis to specify cell fate in Drosophila external sensory organs

During asymmetric cell division in Drosophila sensory organ precursors (SOPs), the Numb protein segregates into one of the two daughter cells, in which it inhibits Notch signalling to specify pIIb cell fate. We show here that Numb acts in SOP cells by inducing the endocytosis of Sanpodo, a four-pass transmembrane protein that has previously been shown to regulate Notch signalling in the central nervous system. In sanpodo mutants, SOP cells divide symmetrically into two pIIb cells. We show that Sanpodo is cortical in pIIa, but colocalizes with Notch and Delta in Rab5- and Rab7-positive endocytic vesicles in pIIb. Sanpodo endocytosis requires alpha-Adaptin, a Numb-binding partner involved in clathrin-mediated endocytosis. In numb or alpha-adaptin mutants, Sanpodo is not endocytosed. Surprisingly, this defect is observed already before and during mitosis, which suggests that Numb not only acts in pIIb, but also regulates endocytosis throughout the cell cycle. Numb binds to Sanpodo by means of its phosphotyrosine-binding domain, a region that is essential for Numb function. Our results establish numb- and alpha-adaptin-dependent endocytosis of Sanpodo as the mechanism by which Notch is regulated during external sensory organ development.     

The identification of 1,3-oxazolidine-2-thiones and 1,3-thiazolidine-2-thiones from the reaction of glucose with benzyl isothiocyanate

The structure of interaction products resulting from the reaction of unmodified glucose with benzyl isothiocyanate is reported. Prior to their identification, the main products of this reaction were isolated using solid-phase extraction (SPE) as well as preparative HPLC. They were then identified by NMR and MS as 3-benzyl-4-hydroxy-5-(D-arabino-1,2,3,4-tetrahydroxybutyl)-1,3-oxazolidine-2-thione, 3-benzyl-4-hydroxy-4-hydroxymethyl-5-(D-erythro-1,2,3-trihydroxypropyl)-1,3-oxazolidine-2-thione, N-benzyl-(D-gluco-4,5-dihydroxy-6-hydroxymethyl-tetrahydropyrano)[2,3-b]oxazolidine-2-thione and 3-benzyl-4-(N-benzyl amino)-5-(D-arabino-1,2,3,4-tetrahydroxybutyl)-1,3-thiazolidine-2-thione. The identity of the last compound was secured by X-ray crystal structure data.     

Mutational and structural studies of the diisopropylfluorophosphatase from Loligo vulgaris shed new light on the catalytic mechanism of the enzyme

The active site, the substrate binding site, and the metal binding sites of the diisopropylfluorophosphatase (DFPase) from Loligo vulgaris have been modified by means of site-directed mutagenesis to improve our understanding of the reaction mechanism. Enzymatic characterization of mutants located in the major groove of the substrate binding pocket indicates that large hydrophobic side chains at these positions are favorable for substrate turnover. Moreover, the active site residue His287 proved to be beneficial, but not essential, for DFP hydrolysis. In most cases, hydrophobic side chains at position 287 led to significant catalytic activities although reduced relative to the wild-type enzyme. With respect to the Ca-1 binding site, where catalysis occurs, various mutants indicated that the net charge at this calcium-binding site as well as the relative positions of the charged calcium ligands is crucial for catalytic activity. The importance of the electrostatic potential at the active site was furthermore revealed by various mutations of residues lining the interior of the central water-filled tunnel, which traverses the entire protein structure. In this respect, the structural features of residue His181, which is located at the opposite end of the DFPase tunnel relative to the active site, were characterized extensively. It was concluded that a tunnel-spanning hydrogen bond network, which includes a large number of apparently slow exchanging water molecules, relays any modifications in the electrostatics of the system to the active site, thus affecting the catalytic reactivity of the enzyme.     

Physiological role of macrophage inflammatory protein-3 alpha induction during maturation of intestinal macrophages

Intestinal macrophages (IMAC) are a central component in the defense of the intestinal mucosa against luminal microbes. In normal mucosa, monocytes differentiate to immunologically tolerant IMAC with a typical phenotype lacking activation markers such as CD14 and TLRs 2 and 4. CD33+ IMAC were isolated from normal intestinal mucosa by immunomagnetic beads. A subtractive hybridization subtracting mRNA from normal IMAC from those of in vitro differentiated macrophages was performed. IMAC differentiation was studied in multicellular spheroids (MCS). Functional assays on migration of CD45R0+ T cells were performed in MCS coculture models. Of 76 clones, 3 obtained by subtractive mRNA hybridization showed >99% homology to mRNA of MIP-3alpha, indicating that this chemokine is induced in IMAC compared with in vitro differentiated macrophages. MIP-3alpha protein expression was confirmed in cryostat sections of normal intestinal mucosa by immunohistochemistry. IMAC in the lamina propria stained positive for MIP-3alpha. FACS of purified IMAC clearly indicated expression of MIP-3alpha in these cells. In the MCS-in vitro differentiation model for IMAC, MIP-3alpha protein expression was absent on day 1 but detectable on day 7 of coculture, demonstrating the induction of MIP-3alpha during differentiation of IMAC. IMAC attracted CD45R0+ T cells to migrate into an MCS coculture model. In human mucosa, a close contact between IMAC and CD45R0+ T cells could be demonstrated. MIP-3alpha is induced during the differentiation of monocytes into IMAC. Our data suggest that MIP-3alpha expression could be involved in the recruitment of CD45R0+ cells into the lamina propria.     

Identification of the tRNA-binding protein Arc1p as a novel target of in vivo biotinylation in Saccharomyces cerevisiae

Biotin is an essential cofactor of cell metabolism serving as a protein-bound coenzyme in ATP-dependent carboxylation, in transcarboxylation, and certain decarboxylation reactions. The involvement of biotinylated proteins in other cellular functions has been suggested occasionally, but available data on this are limited. In the present study, a Saccharomyces cerevisiae protein was identified that reacts with streptavidin on Western blots and is not identical to one of the known biotinylated yeast proteins. After affinity purification on monomeric avidin, the biotinylated protein was identified as Arc1p. Using 14C-labeled biotin, the cofactor was shown to be incorporated into Arc1p by covalent and alkali-stable linkage. Similar to the known carboxylases, Arc1p biotinylation is mediated by the yeast biotin:protein ligase, Bpl1p. Mutational studies revealed that biotinylation occurs at lysine 86 within the N-terminal domain of Arc1p. In contrast to the known carboxylases, however, in vitro biotinylation of Arc1p is incomplete and increases with BPL1 overexpression. In accordance to this fact, Arc1p lacks the canonical consensus sequence of known biotin binding domains, and the bacterial biotin:protein ligase, BirA, is unable to use Arc1p as a substrate. Arc1p was shown previously to organize the association of MetRS and GluRS tRNA synthetases with their cognate tRNAs thereby increasing the substrate affinity and catalytic efficiency of these enzymes. Remarkably, not only biotinylated but also the biotin-free Arc1p obtained by replacement of lysine 86 with arginine were capable of restoring Arc1p function in both arc1Delta and arc1Deltalos1Delta mutants, indicating that biotinylation of Arc1p is not essential for activity.     

ImmunoTrack: the novel antibody-based technology for tracing in animal health

This paper describes a novel antibody-based livestock movement control tool and method of meat allocation, both in livestock husbandry as well as during the meat-processing chain. Immuno Track fulfills diverse prerequisites and meets regulatory demands which are substantial for a successful monitoring technology: (i) the induction of long-lasting antibody responses detectable onsite throughout the whole mast period of pigs, (ii) a single immunization injection with protein derivatives is sufficient to evoke a strong epitope-specific antibody response, and (iii) the complete degradation of the protein markers after the antibody response has been triggered in meatproducing animals such as cattle or pigs. There are diverse fields of application for the Immuno-Track marker technology, such as in quality meat programs, as compliance markers for animal vaccines or as a tool for verification of origin. Combination of this monitoring technology with the husbandry and identification databases for cattle and pigs within the European Community will lead to greater transparency in meat production, thereby regaining consumers' trust in concomitant structures of the meat-producing industry.     

Strategies for the conservation of endangered freshwater pearl mussels (<Emphasis Type="Italic">Margaritifera margaritifera</Emphasis> L.): a synthesis of Conservation Genetics and Ecology

Freshwater pearl mussels (Margartifera margaritifera L.) are among the most critically threatened freshwater bivalves worldwide. The pearl mussel simultaneously fulfils criteria of indicator, flagship, keystone and umbrella species and can thus be considered an ideal target species for the process conservation of aquatic ecosystem functioning. The development of conservation strategies for freshwater pearl mussels and for other bivalve species faces many challenges, including the selection of priority populations for conservation and strategic decisions on habitat restoration and/or captive breeding. This article summarises the current information about the species’ systematics and phylogeny, its distribution and status as well as about its life history strategy and genetic population structure. Based on this information, integrative conservation strategies for freshwater mollusc species which combine genetic and ecological information are discussed. Holistic conservation strategies for pearl mussels require the integration of Conservation Genetics and Conservation Ecology actions at various spatial scales, from the individual and population level to global biodiversity conservation strategies. The availability of high resolution genetic markers for the species and the knowledge of the critical stages in the life cycle, particularly of the most sensitive post-parasitic phase, are important prerequisites for conservation. Effective adaptive conservation management also requires an evaluation of previous actions and management decisions. As with other freshwater bivalves, an integrative conservation approach that identifies and sustains ecological processes and evolutionary lineages is urgently needed to protect and manage freshwater pearl mussel diversity. Such research is important for the conservation of free-living populations, as well as for artificial culturing and breeding techniques, which have recently been or which are currently being established for freshwater pearl mussels in several countries.