Insights into the introduction history and population genetic dynamics of the Argentine black-and-white tegu (Salvator merianae) in Florida.

Invasive species are a major threat to global biodiversity. The US state of Florida is especially susceptible to the spread of exotic reptiles due to its subtropical climate, disturbed habitats, and robust pet trade. The Argentine black-and-white tegu (Salvator merianae) is a large, omnivorous lizard currently established in two different regions of Southern Florida. These two populations pose potential threats to sensitive ground nesting species such as gopher tortoises, American crocodiles, and migratory birds. At present, the introduction histories of these populations and the degree to which they are connected by gene flow are largely unknown. To address these issues, we genotyped S. merianae from Hillsborough and Miami-Dade Counties at ten microsatellite loci to assess intrapopulation genetic diversity, the degree of gene flow between populations, and compare the plausibilities of several potential introduction scenarios. Our results indicate that both populations have low genetic diversity [mean number of effective alleles across loci in both populations = 2.09 and are highly differentiated from each other (GST = 0.170; G″ST = 0.545)]. In addition, our results suggest that these populations underwent a bottleneck event prior to their divergence. We discuss what our results suggest about the histories of Florida's invasive tegu populations, as well as how they inform ongoing management strategies.

Calculation of Relative Binding Free Energy in the Water-Filled Active Site of Oligopeptide-Binding Protein A.

The periplasmic oligopeptide binding protein A (OppA) represents a well-known example of water-mediated protein-ligand interactions. Here, we perform free-energy calculations for three different ligands binding to OppA, using a thermodynamic integration approach. The tripeptide ligands share a high structural similarity (all have the sequence KXK), but their experimentally-determined binding free energies differ remarkably. Thermodynamic cycles were constructed for the ligands, and simulations conducted in the bound and (freely solvated) unbound states. In the unbound state, it was observed that the difference in conformational freedom between alanine and glycine leads to a surprisingly slow convergence, despite their chemical similarity. This could be overcome by increasing the softness parameter during alchemical transformations. Discrepancies remained in the bound state however, when comparing independent simulations of the three ligands. These difficulties could be traced to a slow relaxation of the water network within the active site. Fluctuations in the number of water molecules residing in the binding cavity occur mostly on a timescale larger than the simulation time along the alchemical path. After extensive simulations, relative binding free energies that were converged to within thermal noise could be obtained, which agree well with available experimental data.

A single active site mutation inverts stereoselectivity of 16-hydroxylation of testosterone catalyzed by engineered cytochrome P450 BM3.

Inversion of stereoselectivity: screening of a minimal mutant library revealed a cytochrome P450 BM3 variant M01 A82W S72I capable of producing 16 α-OH-testosterone. Remarkably, a single active site mutation S72I in M01 A82W inverted the stereoselectivity of hydroxylation from 16 ß to 16 α. Introduction of S72I mutation in another 16 ß-OH-selective variant M11 V87I, also resulted in similar inversion of stereoselectivity.

Free energy calculations give insight into the stereoselective hydroxylation of α-ionones by engineered cytochrome P450 BM3 mutants.

Previously, stereoselective hydroxylation of α-ionone by Cytochrome P450 BM3 mutants M01 A82W and M11 L437N was observed. While both mutants hydroxylate α-ionone in a regioselective manner at the C3 position, M01 A82W catalyzes formation of trans-3-OH-α-ionone products whereas M11 L437N exhibits opposite stereoselectivity, producing trans-(3S,6S)-OH-α-ionone and cis-(3S,6R)-OH-α-ionone. Here, we explore the stereoselective C3 hydroxylation of α-ionone by Cytochrome P450 BM3 mutants M01 A82W and M11 L437N using molecular dynamics-based free energy calculations to study the interaction between the enzyme and both the substrates and the products. The one-step perturbation approach is applied using an optimized reference state for substrates and products. While the free energy differences between the substrates free in solution amount to ~0 kJ mol(-1), the differences in mutant M01 A82W agree with the experimentally obtained dissociation constants K(d). Moreover, a correlation with experimentally observed trends in product formation is found in both mutants. The trans isomers show the most favorable relative binding free energy in the range of all four possible hydroxylated diastereomers for mutant M01 A82W, while the trans product from (6S)-α-ionone and the cis product from (6R)-α-ionone show highest affinity for mutant M11 L437N. Marcus theory is subsequently used to relate the thermodynamic stability to transition state energies and rates of formation.

Molecular dynamics simulations and free energy calculations on the enzyme 4-hydroxyphenylpyruvate dioxygenase.

4-Hydroxyphenylpyruvate dioxygenase is a relevant target in both pharmaceutical and agricultural research. We report on molecular dynamics simulations and free energy calculations on this enzyme, in complex with 12 inhibitors for which experimental affinities were determined. We applied the thermodynamic integration approach and the more efficient one-step perturbation. Even though simulations seem well converged and both methods show excellent agreement between them, the correlation with the experimental values remains poor. We investigate the effect of slight modifications on the charge distribution of these highly conjugated systems and find that accurate models can be obtained when using improved force field parameters. This study gives insight into the applicability of free energy methods and current limitations in force field parameterization.

Genetic diversity, admixture, and hatchery influence in Brook Trout (Salvelinus fontinalis) throughout western New York State.

Although Brook Trout are distributed across most of eastern North America, population numbers have declined in many regions due to habitat loss, climate change, and competition with non-native species. In New York State, Brook Trout habitat has been substantially reduced, with many areas showing complete extirpation of Brook Trout populations, predominantly in the western portion of the state. Small, fragmented populations are at risk of genetic diversity loss, inbreeding depression, and reduced fitness, leading to a greater potential for local extirpation. Genetic monitoring is a practical tool that can facilitate further conservation-decision making regarding small populations. In this study, we used 12 microsatellite loci to examine 3,436 sampled Brook Trout, representing 75 sites from the Allegheny, Erie/Niagara, Genesee, Oswego, Lake Ontario, and Susquehanna drainage basins throughout western New York State. Three Brook Trout hatchery strains were also genetically characterized to evaluate the degree of hatchery introgression between wild populations and hatchery strains stocked in the region. Overall, estimates of genetic diversity varied widely: Allelic richness ranged from 2.23 to 7.485, and expected heterozygosity ranged from 0.402 to 0.766. As observed for Brook Trout in other regions, we found a high degree of genetic differentiation among populations, with all comparisons except one showing significant F ST values. Hatchery introgression was found to be minimal, with estimates ranging from 1.96% to 3.10% of wild individuals exhibiting membership proportions to a hatchery strain cluster exceeding 10% (q ≥ 0.10). Results from this investigation can be used to prioritize management efforts for Brook Trout in western New York State and act as a baseline to monitor future population trends.

Structural rationalization of novel drug metabolizing mutants of cytochrome P450 BM3.

Three newly discovered drug metabolizing mutants of cytochrome P450 BM3 (van Vugt-Lussenburg et al., Identification of critical residues in novel drug metabolizing mutants of Cytochrome P450 BM3 using random mutagenesis, J Med Chem 2007;50:455-461) have been studied at an atomistic level to provide structural explanations for a number of their characteristics. In this study, computational methods are combined with experimental techniques. Molecular dynamics simulations, resonance Raman and UV-VIS spectroscopy, as well as coupling efficiency and substrate-binding experiments, have been performed. The computational findings, supported by the experimental results, enable structural rationalizations of the mutants. The substrates used in this study are known to be metabolized by human cytochrome P450 2D6. Interestingly, the major metabolites formed by the P450 BM3 mutants differ from those formed by human cytochrome P450 2D6. The computational findings, supported by resonance Raman data, suggest a conformational change of one of the heme propionate groups. The modeling results furthermore suggest that this conformational change allows for an interaction between the negatively charged carboxylate of the heme substituent and the positively charged nitrogen of the substrates. This allows for an orientation of the substrates favorable for formation of the major metabolite by P450 BM3.

Effect of testosterone on post-myocardial infarction remodeling and function.

BACKGROUND: Men and women are differently affected by coronary artery disease, suggesting an important role of sex steroids. Moreover, testosterone (T) treatment is increasingly used in elderly males. Therefore, we examined effects of chronic anabolic T administration on left ventricular (LV) remodeling after myocardial infarction (MI). METHODS: Adult male rats were treated with intramuscular placebo, testosterone undecanoate (T), or were orchiectomized. After 2 weeks, animals underwent sham-operation (sham) or left coronary artery ligation. Left ventricular remodeling and function was assessed by serial magnetic resonance imaging (MRI) at weeks 2 and 8 and hemodynamic investigation at week 8. RESULTS: In sham operated animals T administration increased serum T levels and led to cardiac hypertrophy, but not to an upregulation of ANP mRNA. The alpha/beta-MHC ratio was significantly higher after T treatment due to an increase in alpha-MHC. As a potential mechanism for this "physiologic" form of hypertrophy, IGF-1 mRNA expression was significantly increased in T treated animals. After coronary artery ligation, infarct size and mortality were similar among the groups. Left ventricular hypertrophy was enhanced by T treatment. However, in vivo LV end-diastolic pressure and wall stress were decreased by T, whereas other hemodynamic parameters (mean arterial pressure, cardiac output, etc.) remained unchanged. CONCLUSION: Chronic anabolic T treatment led to a specific "physiologic" pattern of myocardial hypertrophy with a significant increase in LV weight, but without differences in ANP and with an upregulation in alpha/beta-MHC, possibly mediated by IGF-1. Testosterone treatment had no detrimental effects following MI. Reduced wall stress and LVEDP may even improve long-term outcome.

Administration of testosterone is associated with a reduced susceptibility to myocardial ischemia.

This study investigated the impact of testosterone on myocardial ischemia-reperfusion injury and corresponding intracellular calcium ([Ca2+]i) metabolism. Nonorchiectomized mature male Wistar rats were randomly assigned to placebo, a single dose of testosterone undecanoate, or 5alpha-dihydrotestosterone. In a further series, orchiectomized rats were treated with placebo. After 2 wk of treatment, the hearts were removed and placed in a Langendorff setup. The isolated, buffer-perfused hearts were subjected to 30 min of no-flow ischemia and 30 min of reperfusion. Recovery of myocardial function was measured by analyzing pre- and postischemic left ventricular (LV) systolic/diastolic pressure and coronary perfusion pressure simultaneously, together with [Ca2+]i handling (aequorin luminescence). Calcium regulatory proteins were analyzed by Western blotting. LV weight/body weight ratio was increased after administration of testosterone vs. orchectomized rats. The recovery of contractile function was improved in testosterone-treated rats: at the end of the reperfusion, LV systolic pressure was higher and end-diastolic pressure was lower in testosterone-treated rats. End-ischemic [Ca2+]i and [Ca2+]i overload upon reperfusion was significantly lower in testosterone vs. orchiectomized rats, too. However, levels of calcium regulatory proteins remained unaffected. In conclusion, administration of testosterone significantly improves recovery from global ischemia. These beneficial effects are associated with an attenuation of reperfusion induced [Ca2+]i overload.

The role of protein plasticity in computational rationalization studies on regioselectivity in testosterone hydroxylation by cytochrome P450 BM3 mutants.

Recently, it was found that mutations in the binding cavity of drug-metabolizing Cytochrome P450 BM3 mutants can result in major changes in regioselectivity in testosterone (TES) hydroxylation. In the current work, we report the intrinsic reactivity of TES' C-H bonds and our attempts to rationalize experimentally observed changes in TES hydroxylation using a protein structure-based in silico approach, by setting up and employing a combined Molecular Dynamics (MD) and ligand docking approach to account for the flexibility and plasticity of BM3 mutants. Using this approach, about 100,000 TES binding poses were obtained per mutant. The predicted regioselectivity in TES hydroxylation by the mutants was found to be in disagreement with experiment. As revealed in a detailed structural analysis of the obtained docking poses, this disagreement is due to limitations in correctly scoring hydrogen-bonding and steric interactions with specific active-site residues, which could explain the experimentally observed trends in regioselectivity in TES hydroxylation.

Biophysical and physicochemical methods differentiate highly ligand-efficient human D-amino acid oxidase inhibitors.

Many early drug research efforts are too reductionist thereby not delivering key parameters such as kinetics and thermodynamics of target-ligand binding. A set of human D-Amino Acid Oxidase (DAAO) inhibitors 1-6 was applied to demonstrate the impact of key biophysical techniques and physicochemical methods in the differentiation of chemical entities that cannot be adequately distinguished on the basis of their normalized potency (ligand efficiency) values. The resulting biophysical and physicochemical data were related to relevant pharmacodynamic and pharmacokinetic properties. Surface Plasmon Resonance data indicated prolonged target-ligand residence times for 5 and 6 as compared to 1-4, based on the observed k(off) values. The Isothermal Titration Calorimetry-derived thermodynamic binding profiles of 1-6 to the DAAO enzyme revealed favorable contributions of both ΔH and ΔS to their ΔG values. Surprisingly, the thermodynamic binding profile of 3 elicited a substantially higher favorable contribution of ΔH to ΔG in comparison with the structurally closely related fused bicyclic acid 4. Molecular dynamics simulations and free energy calculations of 1, 3, and 4 led to novel insights into the thermodynamic properties of the binding process at an atomic level and in the different thermodynamic signatures of 3 and 4. The presented holistic approach is anticipated to facilitate the identification of compounds with best-in-class properties at an early research stage.

The role of water molecules in computational drug design.

Although water molecules are small and only consist of two different atom types, they play various roles in cellular systems. This review discusses their influence on the binding process between biomacromolecular targets and small molecule ligands and how this influence can be modeled in computational drug design approaches. Both the structure and the thermodynamics of active site waters will be discussed as these influence the binding process significantly. Structurally conserved waters cannot always be determined experimentally and if observed, it is not clear if they will be replaced upon ligand binding, even if sufficient space is available. Methods to predict the presence of water in protein-ligand complexes will be reviewed. Subsequently, we will discuss methods to include water in computational drug research. Either as an additional factor in automated docking experiments, or explicitly in detailed molecular dynamics simulations, the effect of water on the quality of the simulations is significant, but not easily predicted. The most detailed calculations involve estimates of the free energy contribution of water molecules to protein-ligand complexes. These calculations are computationally demanding, but give insight in the versatility and importance of water in ligand binding.

High-dose 17beta-estradiol treatment prevents development of heart failure post-myocardial infarction in the rat.

OBJECTIVES: Prognosis of heart failure remains poor despite therapeutic advances, such as angiotensin converting enzyme inhibition or beta-receptor blockade. Thus, more effective forms of treatment are urgently needed. Since estrogens have been shown to modulate migration and proliferation of cardiac fibroblasts and to modulate the expression of estrogen receptors of cardiomyocytes we examined whether high-dose estrogen treatment can affect post-myocardial infarction left ventricular remodeling. METHODS: Female rats were treated with 17beta-estradiol (7.5 mg/90 d) or placebo for ten weeks, starting two weeks prior to experimental myocardial infarction. Eight weeks after infarction, in vivo echocardiographic and hemodynamic measurements as well as isolated heart perfusion were performed. RESULTS: In vivo, chronic estrogen treatment almost completely prevented the development of all signs of heart failure that occur in untreated infarcted hearts, such as increased left ventricular diameters (dilatation), reduced fractional shortening (systolic dysfunction) or increased left ventricular end-diastolic pressure (diastolic dysfunction). In vitro, the right- (indicating structural dilatation) and downward (indicating left ventricular dysfunction) shift of left ventricular pressure-volume curves occurring in untreated infarcted hearts was completely prevented by estrogen. CONCLUSIONS: High dose estradiol treatment prevented development of post-MI remodeling, as assessed by in vivo and in vitro parameters of LV dysfunction. Estrogen may hold the potential of becoming a new form of heart failure treatment.However, the mechanisms responsible for this striking and unexpected beneficial action of estrogen in heart failure remain to be elucidated.

Detection of myocardial infarctions by acquisition-weighted 31P-MR spectroscopy in humans.

PURPOSE: To determine whether the recently applied technique of acquisition-weighted 31P-MR spectroscopy (AW-MRS) allows for the detection of depressed energy metabolism in patients with inferior wall myocardial infarctions. MATERIALS AND METHODS: Eight patients with subacute myocardial infarction and wall motion abnormalities restricted to the inferior wall were examined with a 1.5-T MR scanner. Global and regional left ventricular (LV) function was assessed by cine MRI, and the size and extent of myocardial infarction was assessed by late enhancement (LE). MRS was performed with an AW three-dimensional chemical shift imaging sequence. Phosphocreatine/ATP ratios were determined with the postprocessing model AMARES for four voxels positioned in the anterior, lateral, inferior, and septal parts of the LV. RESULTS: The LV ejection fraction (EF) was reduced to 37.5%+/-9.0%. Seven of eight patients had transmural LE in the inferior wall, and one patient showed subendocardial enhancement in the inferior-lateral parts. Phosphocreatine/ATP ratios of the inferior wall were significantly reduced (P <0.05) compared to all other parts of the LV (1.03 +/- 0.39 (inferior), 1.67 +/- 0.81 (lateral), 1.73 +/- 0.29 (anterior), and 1.49 +/- 0.31 (septal)). The ratios in five of seven patients with transmural enhancement were <1.00 in the inferior wall. CONCLUSION: Acquisition weighting allows for the detection of inferior wall infarctions in patients. Transmural signal enhancement is associated with significant depression of phosphocreatine/ATP ratios.

Susceptibility to cardiac ischemia/reperfusion injury is modulated by chronic estrogen status.

The purpose of this study was to test whether the susceptibility of the heart to ischemia/reperfusion injury is modulated by the chronic estrogen status, i.e., increased with estrogen deficiency and attenuated by pharmacologic estrogen supplementation. In addition, the study tested whether estrogen-dependent changes in mechanical function are associated with alterations of cardiac high-energy phosphate metabolism. Rats were ovariectomized, not ovariectomized, or ovariectomized and treated with subcutaneous estrogen pellets (1.5 mg/21 d) (n = 8-11 per group). Three weeks later, hearts were isolated and perfused isovolumically under constant perfusion pressure conditions. Hearts were subjected to 15 min of total global ischemia (37 degrees C) and 30 min of reperfusion. Simultaneous [31P] nuclear magnetic resonance spectra were recorded throughout this protocol to monitor changes in ATP, phosphocreatine, and inorganic phosphate content. Whereas preischemic values for heart rate, end-diastolic pressure, and coronary flow were not different among groups, left ventricular developed pressure was slightly but significantly decreased in the estrogen-treated group (p < 0.05). However, treated hearts showed improved recovery of left ventricular developed pressure on reperfusion (89 +/- 4% in control rats, 70 +/- 8% in ovariectomized hearts, and 114 +/- 9% of preischemic values in estrogen-treated rats). However, changes in ATP, phosphocreatine, and inorganic phosphate during ischemia were as previously described and were unaffected by chronic estrogen status. In conclusion, in the isolated buffer-perfused rat heart, estradiol treatment caused improved functional recovery after ischemia/reperfusion injury. This improvement, however, did not include preservation of high-energy phosphate metabolism. Other potential mechanisms include an anti-oxidant activity of 17beta-estradiol-and estrogen-induced alterations in glucose metabolism.