Compensating bends in a 16-base-pair DNA oligomer containing a T(3)A(3) segment: A NMR study of global DNA curvature

In-phase ligated DNA containing T(n)A(n) segments fail to exhibit the retarded polyacrylamide gel electrophoresis (PAGE) migration observed for in-phase ligated A(n)T(n) segments, a behavior thought to be correlated with macroscopic DNA curvature. The lack of macroscopic curvature in ligated T(n)A(n) segments is thought to be due to cancellation of bending in regions flanking the TpA steps. To address this issue, solution-state NMR, including residual dipolar coupling (RDC) restraints, was used to determine a high-resolution structure of [d(CGAGGTTTAAACCTCG)2], a DNA oligomer containing a T3A3 tract. The overall magnitude and direction of bending, including the regions flanking the central TpA step, was measured using a radius of curvature, Rc, analysis. The Rc for the overall molecule indicated a small magnitude of global bending (Rc = 138 +/- 23 nm) towards the major groove, whereas the Rc for the two halves (72 +/- 33 nm and 69 +/- 14 nm) indicated greater localized bending into the minor groove. The direction of bending in the regions flanking the TpA step is in partial opposition (109 degrees), contributing to cancellation of bending. The cancellation of bending did not correlate with a pattern of roll values at the TpA step, or at the 5' and 3' junctions, of the T3A3 segment, suggesting a simple junction/roll model is insufficient to predict cancellation of DNA bending in all T(n)A(n) junction sequence contexts. Importantly, Rc analysis of structures refined without RDC restraints lacked the precision and accuracy needed to reliably measure bending.     

Bioflavonoid interaction with rat uterine type II binding sites and cell growth inhibition

Competition analysis with a number of known bioflavonoids demonstrated that these compounds (luteolin, quercetin, pelargonin) compete for [3H]estradiol binding to cytosol and nuclear type II sites in rat uterine preparations. The inhibition of [3H]estradiol binding to type II sites was specific and these bioflavonoids did not interact with the rat uterine estrogen receptor. Since estradiol stimulation of nuclear type II sites in the rat uterus is highly correlated with cellular hypertrophy and hyperplasia, we assessed the effects of these compounds on the growth of MCF-7 human breast cancer cells in culture and on estradiol stimulation of uterine growth in the immature rat. The data demonstrated that addition of quercetin (5-10 micrograms/ml) to MCF-7 cell cultures resulted in a dose-dependent inhibition of cell growth (DNA/flask). This effect was reversible by removal of quercetin from the culture medium, or by the addition of 10 nM estradiol-17 beta to these cell cultures containing this bioflavonoid. Since estradiol-17 beta (10 nM) stimulated nuclear type II sites and proliferation of MCF-7 cells, we believe bioflavonoid inhibition of MCF-7 cell growth may be mediated through an interaction with nuclear type II sites. This hypothesis was confirmed by in vivo studies which demonstrated that injection of luteolin or quercetin blocked estradiol stimulation of nuclear type II sites in the immature rat uterus and this correlated with an inhibition of uterine growth (wet and dry weight). These studies suggest bioflavonoids, through an interaction with type II sites, may be involved in cell growth regulation.     

The Bacillus subtilis phage phi 29 protein p16.7, involved in phi 29 DNA replication, is a membrane-localized single-stranded DNA-binding protein.

The functional role of the phi 29-encoded integral membrane protein p16.7 in phage DNA replication was studied using a soluble variant, p16.7A, lacking the N-terminal membrane-spanning domain. Because of the protein-primed mechanism of DNA replication, the bacteriophage phi 29 replication intermediates contain long stretches of single-stranded DNA (ssDNA). Protein p16.7A was found to be an ssDNA-binding protein. In addition, by direct and functional analysis we show that protein p16.7A binds to the stretches of ssDNA of the phi 29 DNA replication intermediates. Properties of protein p16.7A were compared with those of the phi 29-encoded single-stranded DNA-binding protein p5. The results obtained show that both proteins have different, non-overlapping functions. The likely role of p16.7 in attaching phi 29 DNA replication intermediates to the membrane of the infected cell is discussed. Homologues of gene 16.7 are present in phi 29-related phages, suggesting that the proposed role of p16.7 is conserved in this family of phages.     

Effects of City Expansion on Heat Stress under Climate Change Conditions

We examine the joint contribution of urban expansion and climate change on heat stress over the Sydney region. A Regional Climate Model was used to downscale present (1990–2009) and future (2040–2059) simulations from a Global Climate Model. The effects of urban surfaces on local temperature and vapor pressure were included. The role of urban expansion in modulating the climate change signal at local scales was investigated using a human heat-stress index combining temperature and vapor pressure. Urban expansion and climate change leads to increased risk of heat-stress conditions in the Sydney region, with substantially more frequent adverse conditions in urban areas. Impacts are particularly obvious in extreme values; daytime heat-stress impacts are more noticeable in the higher percentiles than in the mean values and the impact at night is more obvious in the lower percentiles than in the mean. Urban expansion enhances heat-stress increases due to climate change at night, but partly compensates its effects during the day. These differences are due to a stronger contribution from vapor pressure deficit during the day and from temperature increases during the night induced by urban surfaces. Our results highlight the inappropriateness of assessing human comfort determined using temperature changes alone and point to the likelihood that impacts of climate change assessed using models that lack urban surfaces probably underestimate future changes in terms of human comfort.     

Resveratrol Ameliorates the Maturation Process of β-Cell-Like Cells Obtained from an Optimized Differentiation Protocol of Human Embryonic Stem Cells

Human embryonic stem cells (hESCs) retain the extraordinary capacity to differentiate into different cell types of an adult organism, including pancreatic β-cells. For this particular lineage, although a lot of effort has been made in the last ten years to achieve an efficient and reproducible differentiation protocol, it was not until recently that this aim was roughly accomplished. Besides, several studies evidenced the impact of resveratrol (RSV) on insulin secretion, even though the mechanism by which this polyphenol potentiates glucose-stimulated insulin secretion (GSIS) is still not clear. The aim of this study was to optimize an efficient differentiation protocol that mimics in vivo pancreatic organogenesis and to investigate whether RSV may improve the final maturation step to obtain functional insulin-secreting cells. Our results indicate that treatment of hESCs (HS-181) with activin-A induced definitive endoderm differentiation as detected by the expression of SOX17 and FOXA2. Addition of retinoic acid (RA), Noggin and Cyclopamine promoted pancreatic differentiation as indicated by the expression of the early pancreatic progenitor markers ISL1, NGN3 and PDX1. Moreover, during maturation in suspension culture, differentiating cells assembled in islet-like clusters, which expressed specific endocrine markers such as PDX1, SST, GCG and INS. Similar results were confirmed with the human induced Pluripotent Stem Cell (hiPSC) line MSUH-001. Finally, differentiation protocols incorporating RSV treatment yielded numerous insulin-positive cells, induced significantly higher PDX1 expression and were able to transiently normalize glycaemia when transplanted in streptozotocin (STZ) induced diabetic mice thus promoting its survival. In conclusion, our strategy allows the efficient differentiation of hESCs into pancreatic endoderm capable of generating β-cell-like cells and demonstrates that RSV improves the maturation process.     

Bone staining in waterlogged deposits: a preliminary contribution to the interpretation of near-shore find accumulation at the Schöningen 13II-4 ‘Spear-Horizon’ site,Lower Saxony,Germany

Abstract

The Schöningen 13II-4 ‘Spear Horizon’ site is famous for the excellent preservation of 300,000-year-old Palaeolithic hunting weapons, including nine wooden spears and a lance, deposited on the shores of a former interglacial lake in association with a large assemblage of well-preserved and butchered animal bones, mainly from horse. Some bones show distinct areas of dark staining, thought to be derived from contact with decaying plant remains along the shores of the lake. It was decided to test this theory and try to determine experimentally where bone staining was most likely to occur on the littoral zone. Modern horse and cow bones were fastened along parallel transects at two locations and the installations were left for several months. Black stains appeared on some bones in the shallows, but not on bones deposited on permanently dry land or in deeper water. Within the 10 m wide band of bones in the main concentration at the Schöningen site, there is a high incidence of bone staining, indicating accumulation of finds along a shallow lake margin. By using GIS, additional clusters of stained bones in the eastern part of the site were revealed and may indicate shorelines when water levels in the lake were lower.     

Structural and Molecular Basis of the Peroxynitrite-mediated Nitration and Inactivation of Trypanosoma cruzi Iron-Superoxide Dismutases (Fe-SODs) A and B: DISPARATE SUSCEPTIBILITIES DUE TO THE REPAIR OF TYR35 RADICAL BY CYS83 IN Fe-SODB THROUGH INTRAMOLECULAR ELECTRON TRANSFER*

Trypanosoma cruzi, the causative agent of Chagas disease, contains exclusively iron-dependent superoxide dismutases (Fe-SODs) located in different subcellular compartments. Peroxynitrite, a key cytotoxic and oxidizing effector biomolecule, reacted with T. cruzi mitochondrial (Fe-SODA) and cytosolic (Fe-SODB) SODs with second order rate constants of 4.6 ± 0.2 × 10⁴ m⁻¹ s⁻¹ and 4.3 ± 0.4 × 10⁴ m⁻¹ s⁻¹ at pH 7.4 and 37 °C, respectively. Both isoforms are dose-dependently nitrated and inactivated by peroxynitrite. Susceptibility of T. cruzi Fe-SODA toward peroxynitrite was similar to that reported previously for Escherichia coli Mn- and Fe-SODs and mammalian Mn-SOD, whereas Fe-SODB was exceptionally resistant to oxidant-mediated inactivation. We report mass spectrometry analysis indicating that peroxynitrite-mediated inactivation of T. cruzi Fe-SODs is due to the site-specific nitration of the critical and universally conserved Tyr³⁵. Searching for structural differences, the crystal structure of Fe-SODA was solved at 2.2 Å resolution. Structural analysis comparing both Fe-SOD isoforms reveals differences in key cysteines and tryptophan residues. Thiol alkylation of Fe-SODB cysteines made the enzyme more susceptible to peroxynitrite. In particular, Cys⁸³ mutation (C83S, absent in Fe-SODA) increased the Fe-SODB sensitivity toward peroxynitrite. Molecular dynamics, electron paramagnetic resonance, and immunospin trapping analysis revealed that Cys⁸³ present in Fe-SODB acts as an electron donor that repairs Tyr³⁵ radical via intramolecular electron transfer, preventing peroxynitrite-dependent nitration and consequent inactivation of Fe-SODB. Parasites exposed to exogenous or endogenous sources of peroxynitrite resulted in nitration and inactivation of Fe-SODA but not Fe-SODB, suggesting that these enzymes play distinctive biological roles during parasite infection of mammalian cells.     

Intraaortic administration of protamine: Method for heparin neutralization after cardiopulmonary bypass

In neutralizing heparin with intravenous protamine sulfate, hypotension may be prevented by administering the drug intraarterially. Forty patients underwent cardiac surgery with extracorporeal circulation in our hospital; each received a rapid injection of nondiluted protamine sulfate in the aortic root to reverse the effects of heparin. To maintain the blood volume at a constant level, volume expanders and inotropic drugs were avoided. The intraaortic injections ranged in duration from 0.2 min to 2.8 min, with a mean of 1.1 min. The mean systolic pressure only dropped from 92 mm Hg (SD +/- 21) before protamine injection to 85 mm Hg (SD +/- 23) after injection (p < 0.0001). In seven patients (18%), no hypotension was evident; in the remaining patients, the systolic pressure returned to preinjection values within a mean of 2.2 min. Coagulation was observed within 3 to 4 min (mean = 2.2 min) after the initiation of injection. This study indicates that intraaortic administration of protamine is a rapid and safe technique for heparin reversal after cardiopulmonary bypass.