Photochemical treatment with endosomally localized photosensitizers enhances the number of adenoviruses in the nucleus

BACKGROUND: In the present study the physical targeting technique photochemical internalization (PCI) has been used in combination with adenovirus. We have previously shown that PCI enhances transgene expression from AdhCMV-lacZ, and the aim of the present study was to further increase the understanding of photochemically mediated adenoviral transduction. METHODS: Two colorectal carcinoma cell lines, WiDr and HCT116, were pre-incubated with the photosensitizer TPPS(2a) or methylene blue derivates (MBD) followed by infection with adenovirus and light exposure. Transgene expression was measured by flow cytometry. Real-time polymerase chain reaction (PCR) and fluorescence in situ hybridization (FISH) were used to quantify the level of viral DNA in the nuclei. Real-time PCR was also used to measure the level of beta-galactosidase mRNA in samples infected with AdhCMV-lacZ. RESULTS: Exposing TPPS(2a)-treated cells to light enhanced the quantity of viral DNA in the nucleus, the mRNA level of the transgene and the transgene expression compared to non-illuminated cells. The increased transgene expression was independent of the promoter used, but dependent on the time of light exposure and the cellular localization of the photosensitizer. CONCLUSIONS: The enhanced transgene expression observed after photochemical treatment is most likely not a result of one event, but more an interplay between various mechanisms. An increased level of adenoviral DNA in the nucleus and a dependency of endosomal localization of the photosensitizer to obtain enhanced transgene expression suggested that endosomal rupture facilitated the transport of adenoviruses to the nucleus.     

Endophytic bacterial communities of field-grown potato plants and their plant-growth-promoting and antagonistic abilities

To study the effect of plant growth on potato-associated bacteria, the composition and properties of bacteria colonizing the endosphere of field-grown potato were analyzed by a multiphasic approach. The occurrence and diversity of potato-associated bacteria were monitored by a cultivation-independent approach, using terminal restriction fragment length polymorphism analysis of 16S rDNA. The patterns obtained revealed a high heterogeneity of community composition and suggested the existence of plant-specific communities. However, endophytic populations correlated to a certain extent with plant growth performance. Endophytes were also isolated from plants that grew well or grew poorly and were identified by partial sequencing of the 16S rRNA genes. A broad phylogenetic spectrum was found among isolates and differently growing plants hosted different bacterial populations. In an approach to investigate the plant-growth-promoting potential of potato-associated bacteria, a total of 35 bacteria were screened by dual testing for in vitro antagonism towards (i) the fungal pathogens Verticillium dahliae, Rhizoctonia solani, Sclerotinia sclerotiorum, and Phytophthora cactorum and (ii) the bacterial pathogens Erwinia carotovora, Streptomyces scabies, and Xanthomonas campestris. The proportion of isolates with antagonistic activity was highest against Streptomyces sp. (43%) followed by those against Xanthomonas sp. (29%). As all plants showed more or less severe disease symptoms of scab disease caused by Streptomyces scabies, we assume that the presence of the pathogen induced the colonization of antagonists. The antifungal activity of the isolates was generally low. The biotechnological potential of endophytic isolates assessed by their antagonistic activity and by in vitro production of enzymes, antibiotics, siderophores, and the plant growth hormone indole-1,3-acetic acid was generally high. Overall, seven endophytes were found to antagonize fungal as well as bacterial pathogens and showed a high production of active compounds and were therefore considered promising biological control agents.     

Mutagenic effect of amniotic fluid from smoking women at term

Concentrated term amniotic fluid samples from 44 women smokers and 44 controls were investigated with respect to mutagenic effect in the Salmonella/mammalian-microsome mutagenicity test, using tester strains TA98 and TA100. Tests with freeze-dried specimens of term amniotic fluid showed increases in the number of revertant colonies over background values, regardless of smoking status. However, samples from heavy smokers produced a higher number of revertants than did samples from nonsmokers in several experiments with tester strain TA98. The increase was statistically significant, using either total tar content or number of cigarettes smoked to identify heavy smokers. Experimental series with tester strain TA100 also resulted in higher group means for heavy smokers than for nonsmokers, but the difference was not statistically significant with the concentrations used in this assay. We conclude that heavy smokers may expose their unborn children to mutagenic substances.     

Molecular cloning and characterization of a nuclear androgen receptor activated by 11-ketotestosterone

Although 11-ketotestosterone is a potent androgen and induces male secondary sex characteristics in many teleosts, androgen receptors with high binding affinity for 11-ketotestosterone or preferential activation by 11-ketotestosterone have not been identified. So, the mechanism by which 11-ketotestosterone exhibits such high potency remains unclear. Recently we cloned the cDNA of an 11-ketotestosterone regulated protein, spiggin, from three-spined stickleback renal tissue. As spiggin is the only identified gene product regulated by 11-ketotestosterone, the stickleback kidney is ideal for determination of the mechanism of 11-ketotestosterone gene regulation. A single androgen receptor gene with two splicing variants, belonging to the androgen receptor-β subfamily was cloned from stickleback kidney. A high affinity, saturable, single class of androgen specific binding sites, with the characteristics of an androgen receptor, was identified in renal cytosolic and nuclear fractions. Measurement of ligand binding moieties in the cytosolic and nuclear fractions as well as to the recombinant receptor revealed lower affinity for 11-ketotestosterone than for dihydrotestosterone. Treatment with different androgens did not up-regulate androgen receptor mRNA level or increase receptor abundance, suggesting that auto-regulation is not involved in differential ligand activation. However, comparison of the trans-activation potential of the stickleback androgen receptor with the human androgen receptor, in both human HepG2 cells and zebrafish ZFL cells, revealed preferential activation by 11-ketotestosterone of the stickleback receptor, but not of the human receptor. These findings demonstrate the presence of a receptor preferentially activated by 11-ketotestosterone in the three-spined stickleback, so far the only one known in any animal.     

Factors contributing to the potency of antimicrobial cationic peptides from the N-terminal region of human lactoferrin

This study investigated the antimicrobial activities of peptides derived from the N-terminal region of human lactoferrin, and examined the contributions of individual residues to the activity of the most potent peptide. Two regions of antimicrobial activity were identified, the first corresponding to a weakly active peptide, HLP-9, comprising residues 1-9, and a second corresponding to a more potent peptide, HLP-10, comprising residues 18-26 and containing the hexapeptide motif, FQWQRN. Inhibitory studies on peptides from the first region confirm the importance of tryptophan residues in enhancing and broadening peptide activity. Inhibitory studies with glycine-substituted homologues of the more potent peptide showed that F21/G and R25/G substitutions resulted in a major reduction or complete loss of activity, while increased peptide cationicity or flexibility had little effect. Our findings demonstrate that F21 and R25 are critical determinants of potency for HLP-10, and that the second aromatic residue may act synergistically with W23 in developing and enhancing the activity of this cationic peptide.     

A practical guide on how to handle patients with bleeding events while on oral antithrombotic treatment

Bleeding is a feared complication in patients who are treated with antithrombotic therapy (oral anticoagulation or antiplatelet therapy). Management of antithrombotic therapy after bleeding poses a dilemma where restarting the crucial medication could lead to recurrent bleeding, while interrupting or even discontinuing treatment could increase the thrombotic risk. In this review, we provide recommendations regarding the treatment of patients with a bleeding event while on oral antithrombotic therapy, based on the literature and expert opinion.     

Context-Dependency of Agricultural Legacies in Temperate Forest Soils

Ecosystems - Anthropogenic activities have affected forests for centuries, leading to persistent legacies. Observations of agricultural legacies on forest soil properties have been site specific...