The effect of divalent metal ions on the electrophoretic mobility of bovine prothrombin and bovine prothrombin fragment 1

Examination of metal ion-dependent effects on the electrophoretic mobility of bovine prothrombin and fragment 1 provides a useful and sensitive method for investigation of conformational processes in these proteins. Utilization of this method reveals a conformational change in bovine prothrombin and fragment 1 which occurs at low metal ion concentrations. Equilibrium dialysis studies indicate that the metal ion-induced shape change occurs concomitant with binding of a single calcium ion/molecule of prothrombin or fragment 1. Mixed metal electrophoretic mobility studies with Mg2+ and Ca2+ have demonstrated the "synergistic" effect for fragment 1 observed by others. Mixed metal equilibrium dialysis has provided experimental support for this observation and allows us to conclude that two tight Ca2+ sites are not affected by low Mg2+ concentrations and that the third Ca2+ site is also a tight site for Mg2+. Thus, at low Mg2+ concentrations and upon the addition of Ca2+, there are effectively three tight sites; consequently more Ca2+ will bind to the protein at lower total Ca2+ ion concentrations.     

Modelling of Thyroid Peroxidase Reveals Insights into Its Enzyme Function and Autoantigenicity

Thyroid peroxidase (TPO) catalyses the biosynthesis of thyroid hormones and is a major autoantigen in Hashimoto’s disease—the most common organ-specific autoimmune disease. Epitope mapping studies have shown that the autoimmune response to TPO is directed mainly at two surface regions on the molecule: immunodominant regions A and B (IDR-A, and IDR-B). TPO has been a major target for structural studies for over 20 years; however, to date, the structure of TPO remains to be determined. We have used a molecular modelling approach to investigate plausible modes of TPO structure and dimer organisation. Sequence features of the C-terminus are consistent with a coiled-coil dimerization motif that most likely anchors the TPO dimer in the apical membrane of thyroid follicular cells. Two contrasting models of TPO were produced, differing in the orientation and exposure of their active sites relative to the membrane. Both models are equally plausible based upon the known enzymatic function of TPO. The “trans” model places IDR-B on the membrane-facing side of the myeloperoxidase (MPO)-like domain, potentially hindering access of autoantibodies, necessitating considerable conformational change, and perhaps even dissociation of the dimer into monomers. IDR-A spans MPO- and CCP-like domains and is relatively fragmented compared to IDR-B, therefore most likely requiring domain rearrangements in order to coalesce into one compact epitope. Less epitope fragmentation and higher solvent accessibility of the “cis” model favours it slightly over the “trans” model. Here, IDR-B clusters towards the surface of the MPO-like domain facing the thyroid follicular lumen preventing steric hindrance of autoantibodies. However, conformational rearrangements may still be necessary to allow full engagement with autoantibodies, with IDR-B on both models being close to the dimer interface. Taken together, the modelling highlights the need to consider the oligomeric state of TPO, its conformational properties, and its proximity to the membrane, when interpreting epitope-mapping data.     

Enzyme electrokinetics: using protein film voltammetry to investigate redox enzymes and their mechanisms

Protein film voltammetry is a relatively new approach to studying redox enzymes, the concept being that a sample of a redox protein is configured as a film on an electrode and probed by a variety of electrochemical techniques. The enzyme molecules are bound at the electrode surface in such a way that there is fast electron transfer and complete retention of the chemistry of the active site that is observed in more conventional experiments. Modulations of the electrode potential or catalytic turnover result in the movement of electrons to, from, and within the enzyme; this is detected as a current that varies in characteristic ways with time and potential. Henceforth, the potential dimension is introduced into enzyme kinetics. The presence of additional intrinsic redox centers for providing fast intramolecular electron transfer between a buried active site and the protein surface is an important factor. Centers which carry out cooperative two-electron transfer, most obviously flavins, produce a particularly sharp signal that allows them to be observed, even as transient states, when spectroscopic methods are not useful. High catalytic activity produces a large amplification of the current, and useful information can be obtained even if the coverage on the electrode is low. Certain enzymes display optimum activity at a particular potential, and this can be both mechanistically informative and physiologically relevant. This paper outlines the principles of protein film voltammetry by discussing some recent results from this laboratory.     

Semi-automated liquid--liquid back-extraction in a 96-well format to decrease sample preparation time for the determination of dextromethorphan and dextrorphan in human plasma

A semi-automated, 96-well based liquid-liquid back-extraction (LLE) procedure was developed and used for sample preparation of dextromethorphan (DEX), an active ingredient in many over-the-counter cough formulations, and dextrorphan (DOR), an active metabolite of DEX, in human plasma. The plasma extracts were analyzed by liquid chromatography-tandem mass spectrometry (LC-MS-MS). The analytes were isolated from human plasma using an initial ether extraction, followed by a back extraction from the ether into a small volume of acidified water. The acidified water isolated from the back extraction was analyzed directly by LC-MS-MS, eliminating the need for a dry down step. A liquid handling system was utilized for all aspects of liquid transfers during the LLE procedure including the transfer of samples from individual tubes into a 96-well format, preparation of standards, addition of internal standard and the addition and transfer of the extraction solvents. The semi-automated, 96-well based LLE procedure reduced sample preparation time by a factor of four versus a comparable manually performed LLE procedure.     

Determination of norepinephrine in small volume plasma samples by stable-isotope dilution gas chromatography–tandem mass spectrometry with negative ion chemical ionization

A stable-isotope based gas chromatography–tandem mass spectrometry–negative ion chemical ionization method was developed for the determination of norepinephrine (NE) levels in small volumes (25–100 μl) of plasma. NE was stabilized in plasma by the addition of semicarbazide and spiked with deuterium-labeled norepinephrine internal standard. The analytes were isolated from the plasma by solid-phase extraction using phenylboronic acid columns and derivatized using pentafluoropropionic anhydride. The derivatized analytes were chromatographed on a capillary column and detected by tandem mass spectrometry with negative ion chemical ionization. Unparalleled sensitivity and selectivity were obtained using this detection scheme, allowing the unambiguous analysis of trace levels of NE in small-volume plasma samples. Linear standard curves were obtained for NE over a mass range from 1 to 200 pg per sample. The method had a limit of quantitation of 10 pg NE/ml plasma when using a 100-μl sample aliquot (1 pg/sample). Accuracy for the analysis of plasma samples spiked with 10 to 200 pg NE/ml typically ranged from 100±10%, with RSD values of less than 10%. The methodology was applied to determine the effect of clonidine on plasma NE levels in conscious spontaneously hypertensive rats. Administration of clonidine (30 μg/kg) produced an 80% reduction in plasma NE accompanied by a 30% reduction in heart and mean arterial pressure that persisted >90 min after drug administration. The ability to take multiple samples from individual rats allowed the time course for the effect of clonidine to be mapped out using only one group of animals.     

Parasitic Absorption and Internal Quantum Efficiency Measurements of Solid‐State Dye Sensitized Solar Cells

The internal quantum efficiency (IQE) of solid‐state dye sensitized solar cells (ssDSCs) is measured using a hybrid optical modeling plus absorptance measurement approach which takes into account the parasitic absorption of the hole transport material (HTM). Across device thicknesses of 1 to 4 microns, ssDSCs sensitized with Z907 and TT1 dyes display relatively constant IQEs of approximately 88% and 36%, respectively, suggesting excellent charge collection efficiencies for both dyes but poor carrier injection for TT1 devices. The addition of more coadsorbent is shown to increase the IQE of TT1 up to approximately 58%, but significantly lowers dye loading. Finally, optical losses due to absorption by the HTM are quantified and found to be a significant contribution to photocurrent losses for ssDSCs sensitized with poor absorbers such as Z907, as the weak absorption of the dye gives the HTM opportunity for significant parasitic absorption within the active layer.     

Sildenafil (Viagra) attenuates ischemic cardiomyopathy and improves left ventricular function in mice

We tested the hypothesis that chronic treatment with sildenafil attenuates myocardial infarction (MI)-induced heart failure. Sildenafil has potent protective effects against necrosis and apoptosis following ischemia-reperfusion in the intact heart and cardiomyocytes. ICR mice underwent MI by left anterior descending coronary artery ligation and were treated with sildenafil (0.71 mg/kg bid) or saline for 4 wk. Infarct size (IS) was measured 24 h postinfarction, and apoptosis was measured by terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling. Left ventricular end-diastolic diameter (LVEDD) and fractional shortening (FS) were measured by echocardiography. Sildenafil reduced IS (40.0 +/- 4.6%) compared with that in saline (69.6 +/- 4.1%, P < 0.05). NG-nitro-l-arginine methyl ester, a nitric oxide synthase (NOS) inhibitor (15 mg/kg bid), blocked the protective effect of sildenafil (IS, 60.2 +/- 1.6%, P < 0.05 vs. sildenafil). Western blot analysis revealed a significant increase in endothelial NOS/inducible NOS proteins 24 h post-MI after treatment with sildenafil versus saline. Apoptosis decreased from 2.4 +/- 0.3% with saline to 1.2 +/- 0.1% with sildenafil (P < 0.05) on day 7 and from 2.0 +/- 0.2% with saline to 1.2 +/- 0.1% with sildenafil on day 28 (P < 0.05), which was associated with an early increase in the Bcl-2-to-Bax ratio. LVEDD increased from baseline value of 3.6 +/- 0.1 to 5.2 +/- 0.2 and to 5.5 +/- 0.1 mm on days 7 and 28, respectively, with saline (P < 0.05) but was attenuated to 4.4 +/- 0.2 and 4.4 +/- 0.1 mm following sildenafil treatment on days 7 and 28, respectively (P > 0.05 vs. baseline). FS significantly improved post-MI with sildenafil. A marked decline in cardiac hypertrophy was observed with sildenafil, which paralleled a reduction in pulmonary edema. Survival rate was lower with saline (36%) compared with sildenafil (93%, P < 0.05). Sildenafil attenuates ischemic cardiomyopathy in mice by limiting necrosis and apoptosis and by preserving left ventricular function possibly through a nitric oxide-dependent pathway.     

The influence of geographic heterogeneity in predation pressure on sexual signal divergence in an Amazonian frog species complex

Sexual selection plays an important role in mating signal divergence, but geographic variation in ecological factors can also contribute to divergent signal evolution. We tested the hypothesis that geographic heterogeneity in predation causes divergent selection on advertisement call complexity within the Engystomops petersi (previously Physalaemus petersi) frog species complex. We conducted predator phonotaxis experiments at two sites where female choice is consistent with call trait divergence. Engystomops at one site produces complex calls, whereas the closely related species at the other site produces simple calls. Bats approached complex calls more than simple calls at both sites, suggesting selection against complex calls. Moreover, bat predation pressure was greater at the site with simple calls, suggesting stronger selection against complex calls and potentially precluding evolution of complex calls at this site. Our results show that geographic variation in predation may play an important role in the evolution and maintenance of mating signal divergence.     

High-latitude<Emphasis Type="Italic"> Acropora cervicornis</Emphasis> thickets off Fort Lauderdale,Florida, USA

Baseline studies conducted in 1998 document the presence of robust, non-reef-building Acropora cervicornis thickets in shallow (3–7 m depth), near-shore waters off the coast of Fort Lauderdale, Florida, USA. These thickets thrive in a high-latitude environment, the northernmost in the continental USA, in the midst of potential anthropogenic stressors. Within thickets, the spatial variation in mean percent coral cover, macroalgal cover, scleractinian species richness, density of A. cervicornis juveniles, and the density and size of A. cervicornis colonies and fragments were recorded. Thicket size ranged between ~0.1 and 0.8 ha and mean coral cover varied between ~5 and 28%, with A. cervicornis accounting for ~87–97% of all scleractinians. Mean A. cervicornis colony and fragment densities per thicket were 1.3–3.3 colonies m^–2 and 0.7–2.8 fragments m^–2, respectively. Recruit densities varied between 0 and 1 ind. m^–2. White band disease was detected at all thickets, with a mean A. cervicornis colony surface area affected of 1.8%. For all thickets, densities of the corallivorous polychaete Hermodice carunculata ranged between ~18 and 86 ind. ha^–1, with predation scars affecting <0.2% of the A. cervicornis cover. These flourishing A. cervicornis thickets off Fort Lauderdale provide an interesting counterpoint to the declining and disease-stricken A. cervicornis populations reported in the Florida Keys and wider Caribbean.