Uptake of heavy metals by plants from airborne deposition and polluted soils

Selostus: Kasvien raskasmetallien otto ilmasta ja saastuneesta maasta

Active uptake of dendritic cell-derived exovesicles by epithelial cells induces the release of inflammatory mediators through a TNF-alpha-mediated pathway.

Dendritic cells (DCs) can release hundreds of membrane vesicles, called exovesicles, which are able to activate resting DCs and distribute antigen. Here, we examined the role of mature DC-derived exovesicles in innate and adaptive immunity, in particular their capacity to activate epithelial cells. Our analysis of exovesicle contents showed that exovesicles contain major histocompatibility complex-II, CD40, and CD83 molecules in addition to tumor necrosis factor (TNF) receptors, TNFRI and TNFRII, and are important carriers of TNF-alpha. These exovesicles are rapidly internalized by epithelial cells, inducing the release of cytokines and chemokines, but do not transfer an alloantigen-presenting capacity to epithelial cells. Part of this activation appears to involve the TNF-alpha-mediated pathway, highlighting the key role of DC-derived exovesicles, not only in adaptive immunity, but also in innate immunity by triggering innate immune responses and activating neighboring epithelial cells to release cytokines and chemokines, thereby amplifying the magnitude of the innate immune response.

Macronutrient uptake, accumulation and export by the irrigated 'vitória' pineapple plant

The nutritional state of the pineapple plant has a large effect on plant growth, on fruit production, and fruit quality. The aim of this study was to assess the uptake, accumulation, and export of nutrients by the irrigated 'Vitória' pineapple plant during and at the end of its development. A randomized block statistical design with four replications was used. The treatments were defined by different times of plant collection: at 270, 330, 390, 450, 510, 570, 690, 750, and 810 days after planting (DAP). The collected plants were separated into the following components: leaves, stem, roots, fruit, and slips for determination of fresh and dry matter weight at 65 ºC. After drying, the plant components were ground for characterization of the composition and content of nutrients taken up and exported by the pineapple plant. The results were subjected to analysis of variance, and non-linear regression models were fitted for the significant differences identified by the F test (p<0.01). The leaves and the stem were the plant components that showed the greatest accumulation of nutrients. For production of 72 t ha-1 of fruit, the macronutrient accumulation in the 'Vitória' pineapple exhibited the following decreasing order: K > N > S > Ca > Mg > P, which corresponded to 898, 452, 134, 129, 126, and 107 kg ha-1, respectively, of total accumulation. The export of macronutrients by the pineapple fruit was in the following decreasing order: K > N > S > Ca > P > Mg, which was equivalent to 18, 17, 11, 8, 8, and 5 %, respectively, of the total accumulated by the pineapple. The 'Vitória' pineapple plant exported 78 kg ha-1 of N, 8 kg ha-1 of P, 164 kg ha-1 of K, 14 kg ha-1 of S, 10 kg ha-1 of Ca, and 6 kg ha-1 of Mg by the fruit. The nutrient content exported by the fruits represent important components of nutrient extraction from the soil, which need to be restored, while the nutrients contained in the leaves, stems and roots can be incorporated in the soil within a program of recycling of crop residues.

Performance of the 47-Kilodalton Membrane Protein versus DNA Polymerase I Genes for Detection of Treponema pallidum by PCR in Ulcers.

Treponema pallidum PCR (Tp-PCR) is a direct diagnostic method for primary and secondary syphilis, but there is no recommendation regarding the best choice of target gene. In this study, we sequentially tested 272 specimens from patients with sexually transmitted ulcers using Tp-PCR targeting the tpp47 and then polA genes. The two methods showed similar accuracies and an almost-perfect agreement.

Binding of double-strand breaks in DNA by human Rad52 protein.

Double-strand breaks (DSBs) in DNA are caused by ionizing radiation. These chromosomal breaks can kill the cell unless repaired efficiently, and inefficient or inappropriate repair can lead to mutation, gene translocation and cancer. Two proteins that participate in the repair of DSBs are Rad52 and Ku: in lower eukaryotes such as yeast, DSBs are repaired by Rad52-dependent homologous recombination, whereas vertebrates repair DSBs primarily by Ku-dependent non-homologous end-joining. The contribution of homologous recombination to vertebrate DSB repair, however, is important. Biochemical studies indicate that Ku binds to DNA ends and facilitates end-joining. Here we show that human Rad52, like Ku, binds directly to DSBs, protects them from exonuclease attack and facilitates end-to-end interactions. A model for repair is proposed in which either Ku or Rad52 binds the DSB. Ku directs DSBs into the non-homologous end-joining repair pathway, whereas Rad52 initiates repair by homologous recombination. Ku and Rad52, therefore, direct entry into alternative pathways for the repair of DNA breaks.

3D reconstruction and comparison of shapes of DNA minicircles observed by cryo-electron microscopy.

We use cryo-electron microscopy to compare 3D shapes of 158 bp long DNA minicircles that differ only in the sequence within an 18 bp block containing either a TATA box or a catabolite activator protein binding site. We present a sorting algorithm that correlates the reconstructed shapes and groups them into distinct categories. We conclude that the presence of the TATA box sequence, which is believed to be easily bent, does not significantly affect the observed shapes.

The Vi element. A transposon-like repeated DNA sequence interspersed in the vitellogenin locus of Xenopus laevis.

A repeated DNA element in Xenopus laevis is described that is present in about 7500 copies dispersed throughout the genome. It was first identified in the 5' flanking region of one vitellogenin gene and was therefore named the Vi element. Seven copies are present within the vitellogenin gene region, three of them within introns of the genes A1, A2 and B2, and the other four copies in the gene flanking regions. Four of these copies have been sequenced. The Vi element is bounded by a well-conserved 13 base-pair inverted repeat; in addition, it is flanked by a three base-pair direct repeat that appears to be site-specific. The length of these four copies varies from 112 to 469 base-pairs; however, sequence homology between the different copies is very high. Their structural characteristics suggest that length heterogeneity may have arisen by either unequal recombinations, deletions or tandem duplications. Altogether, the characteristics and properties of the Vi element indicate that it might represent a mobile genetic element. One of the four copies sequenced is inserted close (position -535) to the transcription initiation site of the vitellogenin gene B2 in a region otherwise showing considerable homology with the closely related gene B1. Nevertheless, the presence of the Vi element does not seem to influence significantly the estrogen-controlled expression of gene B2. In addition, three alleles of this gene created by length polymorphism in intron 3 and in the Vi element inserted near the transcription initiation site are described.

Induction of oxidative stress, lysosome activation and autophagy by nanoparticles in human brain-derived endothelial cells.

Different types of NPs (nanoparticles) are currently under development for diagnostic and therapeutic applications in the biomedical field, yet our knowledge about their possible effects and fate in living cells is still limited. In the present study, we examined the cellular response of human brain-derived endothelial cells to NPs of different size and structure: uncoated and oleic acid-coated iron oxide NPs (8-9 nm core), fluorescent 25 and 50 nm silica NPs, TiO2 NPs (21 nm mean core diameter) and PLGA [poly(lactic-co-glycolic acid)]-PEO [poly(ethylene oxide)] polymeric NPs (150 nm). We evaluated their uptake by the cells, and their localization, generation of oxidative stress and DNA-damaging effects in exposed cells. We show that NPs are internalized by human brain-derived endothelial cells; however, the extent of their intracellular uptake is dependent on the characteristics of the NPs. After their uptake by human brain-derived endothelial cells NPs are transported into the lysosomes of these cells, where they enhance the activation of lysosomal proteases. In brain-derived endothelial cells, NPs induce the production of an oxidative stress after exposure to iron oxide and TiO2 NPs, which is correlated with an increase in DNA strand breaks and defensive mechanisms that ultimately induce an autophagy process in the cells.

Protein-protein interactions between adenovirus DNA polymerase and nuclear factor I mediate formation of the DNA replication preinitiation complex.

The in vitro adenovirus (Ad) DNA replication system provides an assay to study the interaction of viral and host replication proteins with the DNA template in the formation of the preinitiation complex. This initiation system requires in addition to the origin DNA sequences 1) Ad DNA polymerase (Pol), 2) Ad preterminal protein (pTP), the covalent acceptor for protein-primed DNA replication, and 3) nuclear factor I (NFI), a host cell protein identical to the CCAAT box-binding transcription factor. The interactions of these proteins were studied by coimmunoprecipitation and Ad origin DNA binding assays. The Ad Pol can bind to origin sequences only in the presence of another protein which can be either pTP or NFI. While NFI alone can bind to its origin recognition sequence, pTP does not specifically recognize DNA unless Ad Pol is present. Thus, protein-protein interactions are necessary for the targetting of either Ad Pol or pTP to the preinitiation complex. DNA footprinting demonstrated that the Ad DNA site recognized by the pTP.Pol complex was within the first 18 bases at the end of the template which constitutes the minimal origin of replication. Mutagenesis studies have defined the Ad Pol interaction site on NFI between amino acids 68-150, which overlaps the DNA binding and replication activation domain of this factor. A putative zinc finger on the Ad Pol has been mutated to a product that fails to bind the Ad origin sequences but still interacts with pTP. These results indicate that both protein-protein and protein-DNA interactions mediate specific recognition of the replication origin by Ad DNA polymerase.

Alkylpurine-DNA-N-glycosylase confers resistance to temozolomide in xenograft models of glioblastoma multiforme and is associated with poor survival in patients.

Glioblastoma multiforme (GBM) is the most common and lethal of all gliomas. The current standard of care includes surgery followed by concomitant radiation and chemotherapy with the DNA alkylating agent temozolomide (TMZ). O⁶-methylguanine-DNA methyltransferase (MGMT) repairs the most cytotoxic of lesions generated by TMZ, O⁶-methylguanine. Methylation of the MGMT promoter in GBM correlates with increased therapeutic sensitivity to alkylating agent therapy. However, several aspects of TMZ sensitivity are not explained by MGMT promoter methylation. Here, we investigated our hypothesis that the base excision repair enzyme alkylpurine-DNA-N-glycosylase (APNG), which repairs the cytotoxic lesions N³-methyladenine and N⁷-methylguanine, may contribute to TMZ resistance. Silencing of APNG in established and primary TMZ-resistant GBM cell lines endogenously expressing MGMT and APNG attenuated repair of TMZ-induced DNA damage and enhanced apoptosis. Reintroducing expression of APNG in TMZ-sensitive GBM lines conferred resistance to TMZ in vitro and in orthotopic xenograft mouse models. In addition, resistance was enhanced with coexpression of MGMT. Evaluation of APNG protein levels in several clinical datasets demonstrated that in patients, high nuclear APNG expression correlated with poorer overall survival compared with patients lacking APNG expression. Loss of APNG expression in a subset of patients was also associated with increased APNG promoter methylation. Collectively, our data demonstrate that APNG contributes to TMZ resistance in GBM and may be useful in the diagnosis and treatment of the disease.

RNA/DNA co-analysis from blood stains--results of a second collaborative EDNAP exercise.

A second collaborative exercise on RNA/DNA co-analysis for body fluid identification and STR profiling was organized by the European DNA Profiling Group (EDNAP). Six human blood stains, two blood dilution series (5-0.001 μl blood) and, optionally, bona fide or mock casework samples of human or non-human origin were analyzed by the participating laboratories using a RNA/DNA co-extraction or solely RNA extraction method. Two novel mRNA multiplexes were used for the identification of blood: a highly sensitive duplex (HBA, HBB) and a moderately sensitive pentaplex (ALAS2, CD3G, ANK1, SPTB and PBGD). The laboratories used different chemistries and instrumentation. All of the 18 participating laboratories were able to successfully isolate and detect mRNA in dried blood stains. Thirteen laboratories simultaneously extracted RNA and DNA from individual stains and were able to utilize mRNA profiling to confirm the presence of blood and to obtain autosomal STR profiles from the blood stain donors. The positive identification of blood and good quality DNA profiles were also obtained from old and compromised casework samples. The method proved to be reproducible and sensitive using different analysis strategies. The results of this collaborative exercise involving a RNA/DNA co-extraction strategy support the potential use of an mRNA based system for the identification of blood in forensic casework that is compatible with current DNA analysis methodology.