Poly-l-glutamic acid derivatives as multifunctional vectors for gene delivery. Part B. Biological evaluation

Cationic polymers, such as poly-l-lysine (pLL) and polyethyleneimine (pEI), are receiving growing attention as vectors for gene therapy. They form polyelectrolyte complexes with DNA, resulting in a reduced size of the DNA and an enhanced stability toward nucleases. The major disadvantages of using both polymers for in vivo purposes are their cytotoxicity and, in the case of pEI, the fact that it's not biodegradable. In this work, we investigated the interaction between a series of cationic, glutamic acid based polymers and red blood cells. The MTT test was used to investigate the cytotoxicity of the complexes. The ability of the polymers to stabilize DNA toward nucleases was investigated. Transfection studies were carried out on Cos-1 cells. The results from the haemolysis studies, the haemagglutination studies, and the MTT assay show that the polymers are substantially less toxic than pLL and pEI. The polymers are able to protect the DNA from digestion by DNase I. The transfection studies show that the polymer-DNA complexes are capable of transfecting cells, most of them with poor efficiency compared to pEI-DNA complexes.     

The more the merrier: Conspecific density improves performance of gregarious larvae and reduces susceptibility to a pupal parasitoid

Aggregation can confer advantages in animal foraging, defense, and thermoregulation. There is a tight connection between the evolution of insect sociality and a highly effective immune system, presumably to inhibit rapid disease spread in a crowded environment. This connection is less evident for animals that spend only part of their life cycle in a social environment, such as noneusocial gregarious insects. Our aim was to elucidate the effects of group living by the gregarious larvae of the Glanville fritillary butterfly with respect to individual performance, immunity, and susceptibility to a parasitoid. We were also interested in the role of family relative to common postdiapause environment in shaping life‐history traits. Larvae were reared at high or low density and then exposed to the pupal parasitoid wasp Pteromalus apum, either in presence or absence of a previous immune challenge that was used to measure the encapsulation immune response. Surviving adult butterflies were further tested for immunity. The wasp offspring from successfully parasitized butterfly pupae were counted and their brood sex ratios assessed. Larvae reared at high density grew larger and faster than those at low density. Despite high mortality due to parasitism, survival was greater among individuals with high pupal immunity in both density treatments. Moreover, butterfly pupae reared at high density were able to kill a larger fraction of individuals in the parasitoid broods, although this did not increase survival of the host. Finally, a larger proportion of variation observed in most of the traits was explained by butterfly family than by common postdiapause rearing environment, except for adult survival and immunity, for which this pattern was reversed. This gregarious butterfly clearly benefits from high conspecific density in terms of developmental performance and its ability to fight a parasitoid. These positive effects may be driven by cooperative interactions during feeding.     

ClpL is essential for induction of thermotolerance and is potentially part of the HrcA regulon in Lactobacillus gasseri

Stress-inducible proteins are likely to contribute to the survival and activity of probiotic bacteria during industrial processes and in the gastrointestinal tract. The recently published genome sequence of probiotic Lactobacillus gasseri ATCC 33323 suggests the presence of ClpC, ClpE, ClpL, and ClpX from the Clp ATPase family of stress proteins. The heat-shock response of L. gasseri was studied using 2-D DIGE. A total of 20 protein spots showing significant (p<0.05) increase in abundance after 30 min heat-shock were identified, including DnaK, GroEL, ClpC, ClpE, and ClpL. To study the physiological role of ClpL, one of the most highly induced proteins during heat-shock, its corresponding gene was inactivated. The DeltaclpL mutant strain had growth characteristics that were indistinguishable from wild-type under several stress conditions. However, in the absence of functional ClpL, L. gasseri exhibited drastically reduced survival at a lethal temperature and was unable to induce thermotolerance. Genome sequences indicate that the expression of clp genes in several Lactobacillus species is regulated by HrcA, instead of CtsR, the conserved clp gene regulator of low G+C Gram-positive bacteria. Electrophoretic mobility shift assays using L. gasseri HrcA protein and clpL upstream fragments revealed, for the first time, a direct interaction between HrcA and the promoter of a clp gene from a Lactobacillus.     

High-content assay to study protein prenylation

The mevalonate pathway leads to synthesis of cholesterol and isoprenoid lipids. Prenyltransferases attach the isoprenoid lipids to the C-terminus of several small guanosine triphosphate-binding proteins. The prenyl groups are essential for the biological activity of these proteins. The prenyltransferases and other components of the mevalonate pathway are either present or potential drug targets for cancer, osteoporosis, restenosis, or high serum cholesterol level. Until recently, cellular assays to study protein prenylation have been tedious, low-throughput assays. The authors have developed a high-content imaging-based assay to study protein prenylation. The assay is based on a green fluorescent protein (GFP) reporter, which is tagged with the prenylation motif of human H-Ras. The C-terminus of H-Ras targets GFP to the plasma membrane. When protein prenylation is inhibited, the tagged GFP cannot be localized to plasma membrane but is soluble in the cells. The localization of the GFP reporter can be analyzed in the 96- or 384-well format using automated microscopy and automated image analysis. Information about cell number and nuclear intensity can be obtained from the same images. In compound screening, these readouts provide valuable information about the toxicity of the compounds. The authors have validated their assay using several inhibitors of the mevalonate pathway as well as siRNA against farnesyl pyrophosphate synthase, a critical enzyme in the synthesis of the isoprenoid lipids.     

Annexin A2 phosphorylation mediates cell scattering and branching morphogenesis via cofilin Activation

Dynamic remodeling of the actin cytoskeleton is required for cell spreading, motility, and migration and can be regulated by tyrosine kinase activity. Phosphotyrosine proteomic screening revealed phosphorylation of the lipid-, calcium-, and actin-binding protein annexin A2 (AnxA2) at Tyr23 as a major event preceding ts-v-Src kinase-induced cell scattering. Expression of the phospho-mimicking mutant Y23E-AnxA2 itself was sufficient to induce actin reorganization and cell scattering in MDCK cells. While Y23E-AnxA2, but not Y23A-AnxA2, enhanced Src- or hepatocyte growth factor (HGF)-induced cell scattering, short hairpin RNA-mediated knockdown of AnxA2 inhibited both v-Src- and HGF-induced cell scattering. Three-dimensional branching morphogenesis was induced in wild-type-AnxA2-expressing cells only in the presence of HGF, while Y23E-AnxA2 induced HGF-independent branching morphogenesis. Knockdown of AnxA2 prevented lumen formation during cystogenesis. The Y23E-AnxA2-induced scattering was associated with dephosphorylation/activation of the actin-severing protein cofilin. Likewise, inactive S3E-cofilin and constitutively active LIM kinase, a direct upstream kinase of cofilin, inhibited Y23E-AnxA2-induced scattering. Together, our studies indicate an essential role for AnxA2 phosphorylation in regulating cofilin-dependent actin cytoskeletal dynamics in the context of cell scattering and branching morphogenesis.     

Fungal-Mediated Multitrophic Interactions - Do Grass Endophytes in Diet Protect Voles from Predators?

Plant-associated micro-organisms such as mycotoxin-producing endophytes commonly have direct negative effects on herbivores. These effects may be carried over to natural enemies of the herbivores, but this has been rarely explored. We examined how feeding on Neotyphodium endophyte infected (E+) and endophyte free (E−) meadow ryegrass (Scherodonus pratensis) affects body mass, population size and mobility of sibling voles (Microtus levis), and whether the diet mediates the vulnerability of voles to least weasel (Mustela nivalis nivalis) predation. Because least weasels are known to be olfactory hunters, we also examined whether they are able to distinguish olfactory cues of voles fed on E+ and E− diets. Neither body mass of voles nor population size differed between diets. However, contrary to our prediction, least weasels preyed more often on voles fed with E− grass than on voles fed with E+ grass. The mobility of voles fed on E+ grass was reduced compared to voles fed on E− grass, but this effect was unrelated to risk of predation. Least weasels appeared unable to distinguish between excrement odours of voles between the two treatments. Our results suggest that consumption of endophytic grass is not directly deleterious to sibling voles. What''s more, consumption of endophytes appears to be advantageous to voles by reducing risk of mammalian predation. Our study is thus the first to demonstrate an effect of plant-associated microbial symbionts on herbivore-predator interactions in vertebrate communities.     

Potato Glycoalkaloids: A Burden or a Blessing?

Steroidal glycoalkaloids (SGAs) are produced following the general steroid biosynthesis pathway, starting from acetyl-coenzyme A and followed by the intermediates mevalonic acid, squalene, cycloartenol, and cholesterol. α-Chaconine and α-solanine are the main SGAs of the cultivated potato (Solanum tuberosum), whereas many other SGAs are known in the wild potato species. Low concentrations of SGAs improve the taste of potato, but concentrations greater than 200 mg/kg can have toxic effects on animals and humans. SGAs have antimicrobial activity and confer resistance to some insects, but many such pests of potato are not greatly affected. Certain environmental conditions and wounding enhance SGA accumulation in tubers in the field and storage. Low production of SGAs is a dominant character inherited in a relatively simple manner and can be selected for in potato-breeding programs, whereas the use of wild potato germplasm tends to increase the SGA accumulation in the breeding lines. Further efforts are likely to be directed toward the reduction of the SGA content in the edible potato products through breeding and biotechnological methodologies, whereas potato genotypes with high SGA production may be developed for use in the pharmaceutical industry.