Design of native-like proteins through an exposure-dependent environment potential

We hypothesize that the degree of surface exposure of amino acid side chains within a globular, soluble protein has been optimized in evolution, not only to minimize the solvation free energy of the monomeric protein but also to prevent protein aggregation. This effect needs to be taken into account when engineering proteins de novo. We test this hypothesis through addition of a knowledge-based, exposure-dependent energy term to the RosettaDesign solvation potential [Lazaridis, T., and Karplus, M. (1999) Proteins 35, 133-152]. Correlation between amino acid type and surface exposure is determined from a representative set of experimental protein structures. The amino acid solvent accessible surface area (SASA) is estimated with a neighbor vector measure that increases in accuracy compared to the neighbor count measure while remaining pairwise decomposable [Durham, E., et al. (2009) J. Mol. Model. 15, 1093-1108]. Benchmarking of this potential in protein design displays a 3.2% improvement in the overall sequence recovery and an 8.5% improvement in recovery of amino acid types tolerated in evolution.     

Water flux through human aquaporin 1: inhibition by intracellular furosemide and maximal response with high osmotic gradients

This work studies water permeability properties of human aquaporin 1 (hAQP1) expressed in Xenopus laevis oocyte membranes, applying a technique where cellular content is replaced with a known medium, with the possibility of measuring intracellular pressure. Consequences on water transport—produced by well-known anisotonic gradients and by the intracellular effect of probable aquaporin inhibitors—were tested. In this way, the specific intracellular inhibition of hAQP1 by the diuretic drug furosemide was demonstrated. In addition, experiments imposing anisotonic mannitol gradients with a constant ionic strength showed that the relationship between water flux and the applied mannitol gradient deflects from a perfect osmometer response when the gradient is higher than 150 mosmol kg_W⁻¹. These results would indicate that the passage of water molecules through hAQP1 may have a maximum rate. As a whole, this work demonstrates the technical advantage of controlling both intracellular pressure and medium composition in order to study biophysical properties of hAQP1, and contributes information on water channel behavior under osmotic challenges and the discovery of new inhibitors.     

The influence of proximal stem geometry and surface finish on the fixation of a double-tapered cemented femoral stem

In this study, the in vitro fixation of four otherwise identical double-tapered stem-types, varying only in surface finish (polished or matte) and proximal stem geometry (with or without flanges) were compared under two conditions. First, four specimens of each stem type were tested with initially bonded stem–cement interfaces, representing early post-operative conditions. Then, simulating conditions a few weeks to months later, stems were implanted in unused synthetic femurs, with a thin layer coating the stem to prevent stem–cement adhesion. Per-cycle motions were measured at both cement interfaces throughout loading. Overall, surface finish had the smallest relative effect on fixation compared to flanges. Flanges increased axial fixation by 22 μm per-cycle, regardless of surface finish (P=0.01). Further, all stems moved under dynamic load at the stem–cement interface during the first few cycles of loading, even without a thin film. The results indicate that flanges have a greater effect on fixation than surface finish, and therefore adverse findings about matte surfaces should not necessarily apply to all double-tapered stems. Specifically, dorsal flanges enhance the stability of a tapered cemented femoral stem, regardless of surface finish.     

Weeds of Kentucky and Adjacent States. A field guide. By Patricia Dalton Haragan The University Press of Kentucky, Lexington, KY 40506-0336

In 1843 S. B. Buckley (1809–1884) traveled up the St. Johns River, Florida in search of plants, shells, and possibly other natural history objects. Buckley encountered many difficulties in interior Florida and his trip did not live up to his original expectations. It was unfortunate that the took years to distribute his plant specimens, since few botanists or naturalists had preceded him into that poorly explored region.     

Efficacy and Safety of Inhaled Carbon Monoxide during Pulmonary Inflammation in Mice

Background

Pulmonary inflammation is a major contributor to morbidity in a variety of respiratory disorders, but treatment options are limited. Here we investigate the efficacy, safety and mechanism of action of low dose inhaled carbon monoxide (CO) using a mouse model of lipopolysaccharide (LPS)-induced pulmonary inflammation.

Methodology

Mice were exposed to 0–500 ppm inhaled CO for periods of up to 24 hours prior to and following intratracheal instillation of 10 ng LPS. Animals were sacrificed and assessed for intraalveolar neutrophil influx and cytokine levels, flow cytometric determination of neutrophil number and activation in blood, lung and lavage fluid samples, or neutrophil mobilisation from bone marrow.

Principal Findings

When administered for 24 hours both before and after LPS, inhaled CO of 100 ppm or more reduced intraalveolar neutrophil infiltration by 40–50%, although doses above 100 ppm were associated with either high carboxyhemoglobin, weight loss or reduced physical activity. This anti-inflammatory effect of CO did not require pre-exposure before induction of injury. 100 ppm CO exposure attenuated neutrophil sequestration within the pulmonary vasculature as well as LPS-induced neutrophilia at 6 hours after LPS, likely due to abrogation of neutrophil mobilisation from bone marrow. In contrast to such apparently beneficial effects, 100 ppm inhaled CO induced an increase in pulmonary barrier permeability as determined by lavage fluid protein content and translocation of labelled albumin from blood to the alveolar space.

Conclusions

Overall, these data confirm some protective role for inhaled CO during pulmonary inflammation, although this required a dose that produced carboxyhemoglobin values close to potentially toxic levels for humans, and increased lung permeability.     

Pharmacokinetic studies of the herbicide and antitumor compound oryzalin in mice

Oryzalin [3,5-dinitro-N,N-di(n-propyl)benzensulfanilamide] is a widely used sulfonamide herbicide that selectively inhibits microtubule formation in algae and higher plants. Oryzalin has also been found to be an inhibitor of intracellular free Ca²⁺ signalling in mammalian cells and to have antitumor activity in animals. Despite its widespread use there have been no reports of the pharmacokinetics of oryzalin in animals or man. A reversed-phase high-performance liquid chromatographic (HPLC) method was developed to measure oryzalin in biological fluids. Following repeated daily administration of oryzalin to mice by the i.p. route to 200 mg/kg, or the p.o. route at 300 mg/kg, peak plasma concentrations of up to 25 μg/ml were achieved. The half life for oryzalin in plasma of mice given i.p. oryzalin was 14.3 h with a clearance of 0.07 1/h. A major metabolite of oryzalin, N-depropyloryzalin, was identified in plasma and its structure confirmed by mass spectral analysis (M+H⁺ = 305). This metabolite was cleared more rapidly than oryzalin with a half life of 1.15 h and a clearance of 0.17 1/h. N-Depropylorryzalin caused similar inhibition of colony formation by HT-29 colon cancer cells as oryzalin with IC₅₀ = 8 μg/ml. The results suggest that oryzalin and its N-depropyl metabolite can inhibit tumor colony formation at pharmacologically achievable levels.