Effect of arsenic on transcription factor AP-1 and NF-kB DNA binding activity and related gene expression

Both acute (24 h) and chronic (10–20 week) exposure of human fibroblast cells to low dose sodium arsenite (As(III)) significantly affects activating protein-1 (AP-1) and nuclear factor kappa B (NF-κB) DNA binding activity. Short-term treatment with 0.1–5 μM As(III) up-regulates expression of c-Fos and c-Jun and the redox regulators, thioredoxin (Trx) and Redox factor-1 (Ref-1) and activates both AP-1 and NF-κB binding. Chronic exposure to 0.1 or 0.5 μM As(III) decreased c-Jun, c-Fos and Ref-1 protein levels and AP-1 and NF-κB binding activity, but increased Trx expression. Short term exposure to phorbol 12-myristate 13-acetate (TPA), a phorbol ester tumour promoter, or hydrogen peroxide (H₂O₂) also activates AP-1 and NF-κB binding. However, pre-treatment with As(III) prevents this increase. These results suggest that As(III) may alter AP-1 and NF-κB activity, in part, by up-regulating Trx and Ref-1. The different effects of short- versus long-term As(III) treatment on acute-phase response to oxidative stress reflect changes in the expression of Ref-1, c-Fos and c-Jun, but not Trx.

Complete mitochondrial DNA sequences of the decapod crustaceans pseudocarcinus gigas (Menippidae) and macrobrachium rosenbergii (Palaemonidae)

The complete mitochondrial DNA sequence was determined for the Australian giant crab Pseudocarcinns gigas (Crustacea: Decapoda: Menippidae) and the giant freshwater shrimp Macrobrachium rosenbergii (Crustacea: Decapoda: Palaemonidae). The Pse gigas and Mrosenbergii mitochondrial genomes are circular molecules, 15,515 and 15,772 bp in length, respectively, and have the same gene composition as found in other metazoans. The gene arrangement of M. rosenbergii corresponds with that of the presumed ancestral arthropod gene order, represented by Limulus polyphemus, except for the position of the tRNA^Leu(UUR) gene. The Pse. gigas gene arrangement corresponds exactly with that reported for another brachyuran, Portunus trituberculatus, and differs from the M. rosenbergii gene order by only the position of the tRNA^His gene. Given the relative positions of intergenic nonoding nucleotides, the “duplication/random loss” model appears to be the most plausible mechanism for the translocation of this gene. These data represent the first caridean and only the second brachyuran complete mtDNA sequences, and a source of information that will facilitate surveys of intraspecific variation within these commercially important decapod species.

Molecular recognition of DNA by rigid [n]-polynorbornane-derived bifunctional intercalators: synthesis and evaluation of their binding properties

We have exploited the concept of multivalency in the context of DNA recognition, using novel chemistry to synthesize a new type of bis-intercalator with unusual sequence-selectivity. Bis-intercalation has been observed previously, but design principles for de novo construction of such molecules are not known. Our compounds feature two aromatic moieties projecting from a rigid, polynorbornane-based scaffold. The length and character of the backbone as well as the identity of the intercalators were varied, resulting in mono- or divalent recognition of the double helix with varying affinity. Our lead compound proved to be a moderately sequence-selective bis-intercalator with an unwinding angle of 27 and a binding constant of about 8 M. 9-Aminoacridine rings were preferred over acridine carboxamides or naphthalimides, and a rigid [3]-polynorbornane scaffold was superior to a [5]-polynorbornane. The flexibility of the linker connecting the rings to the scaffold, although less influential, could affect the strength and character of the DNA binding.

Intranasal vaccination with proinsulin DNA induces regulatory CD4+ T cells that prevent experimental autoimmune diabetes

Insulin, an autoantigen in type 1 diabetes, when administered mucosally to diabetes-prone NOD mice induces regulatory T cells (T_reg) that protect against diabetes. Compared with protein, Ag encoded as DNA has potential advantages as a therapeutic agent. We found that intranasal vaccination of NOD mice with plasmid DNA encoding mouse proinsulin II-induced CD4⁺ T_reg that suppressed diabetes development, both after adoptive cotransfer with "diabetogenic" spleen cells and after transfer into NOD mice given cyclophosphamide to accelerate diabetes onset. In contrast to prototypic CD4⁺CD25⁺ T_reg, CD4⁺ T_reg induced by proinsulin DNA were both CD25⁺ and CD25^– and not defined by markers such as glucocorticoid-induced TNFR-related protein (GITR), CD103, or Foxp3. Intriguingly, despite induction of T_reg and reduced islet inflammation, diabetes incidence in proinsulin DNA-treated mice was unchanged. However, diabetes was prevented when DNA vaccination was performed under the cover of CD40 ligand blockade, known to prevent priming of CTL by mucosal Ag. Thus, intranasal vaccination with proinsulin DNA has therapeutic potential to prevent diabetes, as demonstrated by induction of protective T_reg, but further modifications are required to improve its efficacy, which could be compromised by concomitant induction of pathogenic immunity.

Colorimetric and fluorescent anion sensors : an overview of recent developments in the use of 1,8-naphthalimide-based chemosensors

This critical review focuses on the development of anion sensors, being either fluorescent and/or colorimetric, based on the use of the 1,8-naphthalimide structure; a highly versatile building unit that absorbs and emits at long wavelengths. The review commences with a short description of the most commonly used design principles employed in chemosensors, followed by a discussion on the photophysical properties of the 4-amino-1,8-naphthalimide structure which has been most commonly employed in both cation and anion sensing to date. This is followed by a review of the current state of the art in naphthalimide-based anion sensing, where systems using ureas, thioureas and amides as hydrogen-bonding receptors, as well as charged receptors have been used for anion sensing in both organic and aqueous solutions, or within various polymeric networks, such as hydrogels. The review concludes with some current and future perspectives including the use of the naphthalimides for sensing small biomolecules, such as amino acids, as well as probes for incorporation and binding to proteins; and for the recognition/sensing of polyanions such as DNA, and their potential use as novel therapeutic and diagnostic agents (95 references).

8-modified-2'-deoxyadenosine analogues induce delayed polymerization arrest during HIV-1 reverse transcription

The occurrence of resistant viruses to any of the anti-HIV-1 compounds used in the current therapies against AIDS underlies the urge for the development of new drug targets and/or new drugs acting through novel mechanisms. While all anti-HIV-1 nucleoside analogues in clinical use and in clinical trials rely on ribose modifications for activity, we designed nucleosides with a natural deoxyribose moiety and modifications of position 8 of the adenine base. Such modifications might induce a steric clash with helix αH in the thumb domain of the p66 subunit of HIV-1 RT at a distance from the catalytic site, causing delayed chain termination. Eleven new 2′-deoxyadenosine analogues modified on position 8 of the purine base were synthesized and tested in vitro and in cell-based assays. In this paper we demonstrate for the first time that chemical modifications on position 8 of 2′-deoxyadenosine induce delayed chain termination in vitro, and also inhibit DNA synthesis when incorporated in a DNA template strand. Furthermore, one of them had moderate anti-HIV-1 activity in cell-culture. Our results constitute a proof of concept indicating that modification on the base moiety of nucleosides can induce delayed polymerization arrest and inhibit HIV-1 replication.

Extracting DNA from museum bird eggs, and whole genome amplification of archive DNA

We present a comprehensive protocol for extracting DNA from egg membranes and other internal debris recovered from the interior of blown museum bird eggs. A variety of commercially available DNA extraction methods were found to be applicable. DNA sequencing of polymerase chain reaction (PCR) products for a 176-bp fragment of mitochondrial DNA was successful for most egg samples (> 78%) even though the amount of DNA extracted (mean = 14.71 ± 4.55 ng/µL) was significantly less than that obtained for bird skin samples (mean = 67.88 ± 4.77 ng/µL). For PCR and sequencing of snipe (Gallinago) DNA, we provide eight new primers for the ‘DNA barcode’ region of COI mtDNA. In various combinations, the primers target a range of PCR products sized from 72 bp to the full ‘barcode’ of 751 bp. Not all possible combinations were tested with archive snipe DNA, but we found a significantly better success rate of PCR amplification for a shorter 176-bp target compared with a larger 288-bp fragment (67% vs. 39%). Finally, we explored the feasibility of whole genome amplification (WGA) for extending the use of archive DNA in PCR and sequencing applications. Of two WGA approaches, a PCR-based method was found to be able to amplify whole genomic DNA from archive skins and eggs from museum bird collections. After WGA, significantly more archive egg samples produced visible PCR products on agarose (56.9% before WGA vs. 79.0% after WGA). However, overall sequencing success did not improve significantly (78.8% compared with 83.0%).

Selection of DNA aptamers against epithelial cell adhesion molecule for cancer cell imaging and circulating tumor cell capture

Epithelial cell adhesion molecule (EpCAM) is overexpressed in most solid cancers and is an ideal antigen for clinical applications in cancer diagnosis, prognosis, imaging, and therapy. Currently, most of the EpCAM-based diagnostic, prognostic, and therapeutic strategies rely on the anti-EpCAM antibody. However, the use of EpCAM antibody is restricted due to its large size and instability. In this study, we have successfully identified DNA aptamers that selectively bind human recombinant EpCAM protein. The aptamers can specifically recognize a number of live human cancer cells derived from breast, colorectal, and gastric cancers that express EpCAM but not bind to EpCAM-negative cells. Among the aptamer sequences identified, a hairpin-structured sequence SYL3 was optimized in length, resulting in aptamer sequence SYL3C. The Kd values of the SYL3C aptamer against breast cancer cell line MDA-MB-231 and gastric cancer cell line Kato III were found to be 38±9 and 67±8 nM, respectively, which are better than that of the full-length SYL3 aptamer. Flow cytometry analysis results indicated that the SYL3C aptamer was able to recognize target cancer cells from mixed cells in cell media. When used to capture cancer cells, up to 63% cancer cell capture efficiency was achieved with about 80% purity. With the advantages of small size, easy synthesis, good stability, high binding affinity, and selectivity, the DNA aptamers reported here against cancer biomarker EpCAM will facilitate the development of novel targeted cancer therapy, cancer cell imaging, and circulating tumor cell detection.

Folic acid supplementation in postpolypectomy patients in a randomized controlled trial increases tissue folate concentrations and reduces aberrant DNA biomarkers in colonic tissues adjacent to the former polyp site

BACKGROUND: Low folate status is associated with an increased risk of colorectal carcinogenesis. Optimal folate status may be genoprotective by preventing uracil misincorporation into DNA and DNA hypomethylation. Adenomatous polyps have low folate status compared with normal colonic mucosa, and they are surrounded by histologically normal mucosa that also is of low folate status. OBJECTIVE: In a randomized controlled trial conducted at a single Dublin hospital between April 2002 and March 2004, we assessed the effect of folic acid supplementation on tissue folate, uracil misincorporation into DNA, and global DNA hypomethylation in colonocytes isolated from sites of adenomatous polyps and from histologically normal tissue adjacent and 10-15 cm distal to them. METHODS: Twenty patients with adenomatous polyps on initial colonoscopy and polypectomy were randomly assigned to receive either 600 μg folic acid/d [n = 12, 38% men, mean age 64.3 y, and body mass index (BMI, in kg/m(2)) 26.6] or placebo (n = 8, 50% men, mean age 68.4 y, and BMI 27.2) for 6 mo, and then repeat the colonoscopy. Blood and colonocyte tissue folate concentrations were measured with the use of a microbiological assay. Uracil misincorporation and global DNA hypomethylation were measured in colonocytes with the use of modified comet assays. RESULTS: Over time, folic acid supplementation, compared with placebo, increased tissue folate (mean ± SEM) from 15.6 ± 2.62 pg/10(5) cells to 18.1 ± 2.12 pg/10(5) cells (P < 0.001) and decreased the global DNA hypomethylation ratio from 1.7 ± 0.1 to 1.0 ± 0.1 (P < 0.001). The uracil misincorporation ratio decreased by 0.5 ± 0.1 for the site adjacent to the polyp over time (P = 0.05). CONCLUSION: A response to folic acid supplementation, which increased colonocyte folate and improved folate-related DNA biomarkers of cancer risk, was seen in the participants studied. Exploratory analysis points toward the area formerly adjacent to polyps as possibly driving the response. That these areas persist after polypectomy in the absence of folate supplementation is consistent with a potentially carcinogenic field's causing the appearance of the polyp.