AN IMPROVED SYNTHESIS OF 9-[2-(DIETHOXYPHOSPHONOMETHOXY)ETHYL]ADENINE AND ITS ANALOGUES WITH OTHER PURINE BASES UTILIZING THE MITSUNOBU REACTION

Abstract

Unprotected adenine, 6-chloropurine, 2.6-diaminopurine. and 2-amino-6-chluropurine have been directly coupled with 2-(diethoxyphosphonomethoxy)ethanol under Mitsunobu reaction conditions to provide acyclic phosphonate nucleotide analogues which are intermediates for antiviral agents such as PMEA.     

Comparison of laboratory and field testing performance evaluations of siloxane-polyurethane fouling-release marine coatings

A series of eight novel siloxane-polyurethane fouling-release (FR) coatings were assessed for their FR performance in both the laboratory and in the field. Laboratory analysis included adhesion assessments of bacteria, microalgae, macroalgal spores, adult barnacles and pseudobarnacles using high-throughput screening techniques, while field evaluations were conducted in accordance with standardized testing methods at three different ocean testing sites over the course of six-months exposure. The data collected were subjected to statistical analysis in order to identify potential correlations. In general, there was good agreement between the laboratory screening assays and the field assessments, with both regimes clearly distinguishing the siloxane-polyurethane compositions comprising monofunctional poly(dimethyl siloxane) (PDMS) (m-PDMS) as possessing superior, broad-spectrum FR properties compared to those prepared with difunctional PDMS (d-PDMS). Of the seven laboratory screening techniques, the Cellulophaga lytica biofilm retraction and reattached barnacle (Amphibalanus amphitrite) adhesion assays were shown to be the most predictive of broad-spectrum field performance.     

Seasonal nutritional status in Norway lobsters,Nephrops norvegicus (L.): are females nutritionally compromised over the winter?

Norway lobsters, Nephrops norvegicus, are sediment-dwelling decapod crustaceans that excavate burrows from which they make short excursions to feed by predation and scavenging. The females of this species are known to reside within their burrows for an extended period of time over the winter while brooding their eggs. The aim of this study was to assess the likelihood of these females being able to feed during this brooding period. Biophysical and biochemical measurements that had previously been shown to change with starvation under laboratory conditions in male N. norvegicus were taken for female N. norvegicus under similar conditions. These measurements were also compared in both sexes obtained from monthly trawl samples from the Clyde Sea Area, Scotland, UK, together with trawl composition data. The laboratory study showed that the hepatosomatic index, and the copper, lipid and water content of the hepatopancreas can be used as indicators of the state of starvation in females, as in males. In the wild, both sexes have reduced nutritional status during the winter, but not to the degree seen in animals starved for 20 weeks in aquarium trials. This study does not support the hypothesis that females cease feeding over winter, during their brooding period. Firstly, some females were unable to sustain ovary development during starvation under controlled conditions, contrary to field observations. Secondly, field data suggest that there is no sex-specific reduction in nutritional status.     

Genetic Variants in the Bone Morphogenic Protein Gene Family Modify the Association between Residential Exposure to Traffic and Peripheral Arterial Disease

There is a growing literature indicating that genetic variants modify many of the associations between environmental exposures and clinical outcomes, potentially by increasing susceptibility to these exposures. However, genome-scale investigations of these interactions have been rarely performed particularly in the case of air pollution exposures. We performed race-stratified genome-wide gene-environment interaction association studies on European-American (EA, N = 1623) and African-American (AA, N = 554) cohorts to investigate the joint influence of common single nucleotide polymorphisms (SNPs) and residential exposure to traffic (“traffic exposure”)—a recognized vascular disease risk factor—on peripheral arterial disease (PAD). Traffic exposure was estimated via the distance from the primary residence to the nearest major roadway, defined as the nearest limited access highways or major arterial. The rs755249-traffic exposure interaction was associated with PAD at a genome-wide significant level (P = 2.29x10^-8) in European-Americans. Rs755249 is located in the 3’ untranslated region of BMP8A, a member of the bone morphogenic protein (BMP) gene family. Further investigation revealed several variants in BMP genes associated with PAD via an interaction with traffic exposure in both the EA and AA cohorts; this included interactions with non-synonymous variants in BMP2, which is regulated by air pollution exposure. The BMP family of genes is linked to vascular growth and calcification and is a novel gene family for the study of PAD pathophysiology. Further investigation of BMP8A using the Genotype Tissue Expression Database revealed multiple variants with nominally significant (P < 0.05) interaction P-values in our EA cohort were significant BMP8A eQTLs in tissue types highlight relevant for PAD such as rs755249 (tibial nerve, eQTL P = 3.6x10^-6) and rs1180341 (tibial artery, eQTL P = 5.3x10^-6). Together these results reveal a novel gene, and possibly gene family, associated with PAD via an interaction with traffic air pollution exposure. These results also highlight the potential for interactions studies, particularly at the genome scale, to reveal novel biology linking environmental exposures to clinical outcomes.     

Characterization of Tiki,a New Family of Wnt-specific Metalloproteases

The Wnt family of secreted glycolipoproteins plays pivotal roles in development and human diseases. Tiki family proteins were identified as novel Wnt inhibitors that act by cleaving the Wnt amino-terminal region to inactivate specific Wnt ligands. Tiki represents a new metalloprotease family that is dependent on Mn²⁺/Co²⁺ but lacks known metalloprotease motifs. The Tiki extracellular domain shares homology with bacterial TraB/PrgY proteins, known for their roles in the inhibition of mating pheromones. The TIKI/TraB fold is predicted to be distantly related to structures of additional bacterial proteins and may use a core β-sheet within an α+β-fold to coordinate conserved residues for catalysis. In this study, using assays for Wnt3a cleavage and signaling inhibition, we performed mutagenesis analyses of human TIKI2 to examine the structural prediction and identify the active site residues. We also established an in vitro assay for TIKI2 protease activity using FRET peptide substrates derived from the cleavage motifs of Wnt3a and Xenopus wnt8 (Xwnt8). We further identified two pairs of potential disulfide bonds that reside outside the β-sheet catalytic core but likely assist the folding of the TIKI domain. Finally, we systematically analyzed TIKI2 cleavage of the 19 human WNT proteins, of which we identified 10 as potential TIKI2 substrates, revealing the hydrophobic nature of Tiki cleavage sites. Our study provides insights into the Tiki family of proteases and its Wnt substrates.     

Increased Frequency of T Follicular Helper Cells and Elevated Interleukin-27 Plasma Levels in Patients with Pemphigus

Pemphigus is an autoimmune disease in which IgG auto-antibodies (auto-ab) against the desmosomal cadherins desmoglein (Dsg) 3 and Dsg1 cause loss of epidermal keratinocyte adhesion. Aim of this study was to investigate cytokines derived from antigen-presenting cells (APC) and their relation to CD4⁺ T cell subpopulations and to the auto-ab response in pemphigus. In this regard, patients with pemphigus were compared to patients with myasthenia gravis (MG), an unrelated auto-ab–mediated autoimmune disease, and healthy controls. In pemphigus and MG, the plasma concentrations of the APC-derived immunomodulatory cytokine IL-27 were highly increased. Strikingly, IL-27 strongly correlated with Dsg-specific IgG auto-ab titers. T helper (Th) 17 cells were augmented in both pemphigus and MG patients while T follicular helper (Tfh) cells, which are essential in providing B cell help, were increased only in pemphigus along with increasing plasma concentrations of IL-21, a cytokine produced by Th17 and Tfh cells. Moreover, we could detect Dsg3-specific autoreactive T cells producing IL-21 upon ex vivo stimulation with Dsg3. These findings suggest that IL-27 and IL-21-producing T cells, are involved in the pathogenesis of pemphigus. The further characterization of IL-21-producing T cells and of the role of IL-27 will lead to a more defined understanding of the auto-ab response in pemphigus.     

Demonstration of Cholinesterase in Teeth

Human teeth have been studied by treatment with copper thio-choline, the method developed by Koelle for demonstrating activity of both specific and nonspecific cholinesterases. Freshly extracted teeth were collected and immediately sectioned on a cutting machine designed for calcified tissues. One series of teeth was sectioned sufficiently thin for microscopic study. Another series of teeth was bisected to expose the pulp chambers to the reagents. These teeth were divided into 5 experimental groups. The first group was treated with 10^-6M di-isopropylfluorophosphate (DFP) for 30 min at 37°C and then incubated with acetylthiocholine (AThCh) for 16 to 20 hr at 37°C in order to reveal the sites of activity of the specific enzyme, AChEase. The second group was incubated in a substrate of butyrylthiocholine (BuThCh) for 12 to 16 hr at 37°C to indicate the sites of the nonspecific ChEase. The third group was incubated in AThCh for 16 to 20 hr at 37°C without previous treatment by an inhibitor in order to reveal the presence and location of both the specific and nonspecific ChEase. The fourth and fifth groups were utilized as controls. Group 4 tissues were incubated without a substrate while those of group 5 were treated with DFP and then incubated with BuThCh. The specimens then were treated with ammonium sulfide to outline the sites of ChEase activity. The thin sections were mounted directly but the series of halved teeth were decalcified, embedded in paraffin, sectioned and then mounted. By these methods the presence and location of both specific and nonspecific ChEase activity were observed in human teeth. Concentration of specific ChEase was observed along the coronal aspect of the pulp chamber and along the course of the pulpal nerves. The nonspecific ChEase was observed throughout the pulpal tissue and appeared to be concentrated along the nerves and blood vessels. Neither series of control tissues exhibited any staining in the pulp tissue.