Influence of mutations of Val226 on the catalytic rate of haloalkane dehalogenase

Haloalkane dehalogenase converts haloalkanes to their corresponding alcohols. The 3D structure, reaction mechanism and kinetic mechanism have been studied. The steady state kcat with 1,2-dichloroethane and 1,2-dibromoethane is limited mainly by the rate of release of the halide ion from the buried active-site cavity. During catalysis, the halogen that is cleaved off (Clα) from 1,2-dichloroethane interacts with Trp125 and the Clβ interacts with Phe172. Both these residues have van der Waals contacts with Val226. To establish the effect of these interactions on catalysis, and in an attempt to change enzyme activity without directly mutating residues involved in catalysis, we mutated Val226 to Gly, Ala and Leu. The Val226Ala and Val226Leu mutants had a 2.5-fold higher catalytic rate for 1,2-dibromoethane than the wild-type enzyme. A pre-steady state kinetic analysis of the Val226Ala mutant enzyme showed that the increase in kcat could be attributed to an increase in the rate of a conformational change that precedes halide release, causing a faster overall rate of halide dissociation. The kcat for 1,2-dichloroethane conversion was not elevated, although the rate of chloride release was also faster than in the wild-type enzyme. This was caused by a 3-fold decrease in the rate of formation of the alkyl-enzyme intermediate for 1,2-dichloroethane. Val226 seems to contribute to leaving group (Clα or Brα) stabilization via Trp125, and can influence halide release and substrate binding via an interaction with Phe172. These studies indicate that wild-type haloalkane dehalogenase is optimized for 1,2-dichloroethane, although 1,2-dibromoethane is a better substrate.

Hypertensive Disorders of Pregnancy and Future Cardiovascular Health.

Hypertensive disorders of pregnancy (HDP) occur in almost 10% of gestations. These women are known to have higher cardiovascular morbidity and mortality later in life in comparison with parous controls who had normotensive pregnancies. Several studies have demonstrated that women with preeclampsia present in a state of segmental impaired myocardial function, biventricular chamber dysfunction, adverse biventricular remodeling, and hypertrophy, a compromised hemodynamic state and indirect echocardiographic signs of localized myocardial ischemia and fibrosis. These cardiac functional and geometric changes are known to have strong predictive value for cardiovascular disease in non-pregnant subjects. A "dose effect" response seems to regulate this relationship with severe HDP, early-onset HDP, coexistence of fetal growth disorders, and recurrence of HDP resulting in poorer cardiovascular measures. The mechanism underlying the relationship between HDP in younger women and cardiovascular disease later in life is unclear but could be explained by sharing of pre-pregnancy cardiovascular risk factors or due to a direct impact of HDP on the maternal cardiovascular system conferring a state of increased susceptibility to future metabolic or hemodynamic insults. If so, the prevention of HDP itself would become all the more urgent. Shortly after delivery, women who experienced HDP express an increased risk of classic cardiovascular risk factors such as essential hypertension, renal disease, abnormal lipid profile, and diabetes with higher frequency than controls. Within one or two decades after delivery, this group of women are more likely to experience premature cardiovascular events, such as symptomatic heart failure, myocardial ischemia, and cerebral vascular disease. Although there is general agreement that women who suffered from HDP should undertake early screening for cardiovascular risk factors in order to allow for appropriate prevention, the exact timing and modality of screening has not been standardized yet. Our findings suggest that prevention should start as early as possible after delivery by making the women aware of their increased cardiovascular risk and encouraging weight control, stop smoking, healthy diet, and daily exercise which are well-established and cost-effective prevention strategies

Preeclampsia: The Relationship between Uterine Artery Blood Flow and Trophoblast Function.

Maternal uterine artery blood flow is critical to maintaining the intrauterine environment, permitting normal placental function, and supporting fetal growth. It has long been believed that inadequate transformation of the maternal uterine vasculature is a consequence of primary defective trophoblast invasion and leads to the development of preeclampsia. That early pregnancy maternal uterine artery perfusion is strongly associated with placental cellular function and behaviour has always been interpreted in this context. Consistently observed changes in pre-conceptual maternal and uterine artery blood flow, abdominal pregnancy implantation, and late pregnancy have been challenging this concept, and suggest that abnormal placental perfusion may result in trophoblast impairment, rather than the other way round. This review focuses on evidence that maternal cardiovascular function plays a significant role in the pathophysiology of preeclampsia

Antenna subtraction with massive fermions at NNLO: Double real initial-final configurations

We derive the integrated forms of specific initial-final tree-level four-parton antenna functions involving a massless initial-state parton and a massive final-state fermion as hard radiators. These antennae are needed in the subtraction terms required to evaluate the double real corrections to $t\bar{t}$ hadronic production at the NNLO level stemming from the partonic processes $q\bar{q}\to t\bar{t}q'\bar{q}'$ and $gg\to t\bar{t}q\bar{q}$.Comment: 24 pages, 1 figure, 1 Mathematica file attache

Kinetic Characterization and X-ray Structure of a Mutant of Haloalkane Dehalogenase with Higher Catalytic Activity and Modified Substrate Range

Conversion of halogenated aliphatics by haloalkane dehalogenase proceeds via the formation of a covalent alkyl-enzyme intermediate which is subsequently hydrolyzed by water. In the wild type enzyme, the slowest step for both 1,2-dichloroethane and 1,2-dibromoethane conversion is a unimolecular enzyme isomerization preceding rapid halide dissociation. Phenylalanine 172 is located in a helix-loop-helix structure that covers the active site cavity of the enzyme, interacts with the Clβ of 1,2-dichloroethane during catalysis, and could be involved in stabilization of this helix-loop-helix region of the cap domain of the enzyme. To obtain more information about the role of this residue in dehalogenase function, we performed a mutational analysis of position 172 and studied the kinetics and X-ray structure of the Phe172Trp enzyme. The Phe172Trp mutant had a 10-fold higher kcat/Km for 1-chlorohexane and a 2-fold higher kcat for 1,2-dibromoethane than the wild-type enzyme. The X-ray structure of the Phe172Trp enzyme showed a local conformational change in the helix-loop-helix region that covers the active site. This could explain the elevated activity for 1-chlorohexane of the Phe172Trp enzyme, since it allows this large substrate to bind more easily in the active site cavity. Pre-steady-state kinetic analysis showed that the increase in kcat found for 1,2-dibromoethane conversion could be attributed to an increase in the rate of an enzyme isomerization step that preceeds halide release. The observed conformational difference between the helix-loop-helix structures of the wild-type enzyme and the faster mutant suggests that the isomerization required for halide release could be a conformational change that takes place in this region of the cap domain of the dehalogenase. It is proposed that Phe172 is involved in stabilization of the helix-loop-helix structure that covers the active site of the enzyme and creates a rigid hydrophobic cavity for small apolar halogenated alkanes.

Gating-by-tilt of mechanosensitive membrane channels

We propose an alternative mechanism for the gating of biological membrane channels in response to membrane tension that involves a change in the slope of the membrane near the channel. Under biological membrane tensions we show that the energy difference between the closed (tilted) and open (untilted) states can far exceed kBT and is comparable to what is available under simple ilational gating. Recent experiments demonstrate that membrane leaflet asymmetries (spontaneous curvature) can strong effect the gating of some channels. Such a phenomenon would be more easy to explain under gating-by-tilt, given its novel intrinsic sensitivity to such asymmetry.Comment: 10 pages, 2 figure

Assessing the impact of intervention strategies against Taenia solium cysticercosis using the EPICYST transmission model

Background The pork tapeworm, Taenia solium, and associated human infections, taeniasis, cysticercosis and neurocysticercosis, are serious public health problems, especially in developing countries. The World Health Organization (WHO) has set goals for having a validated strategy for control and elimination of T. solium taeniasis/cysticercosis by 2015 and interventions scaled-up in selected countries by 2020. Timely achievement of these internationally-endorsed targets requires that the relative benefits and effectiveness of potential interventions be explored rigorously within a quantitative framework. Methods A deterministic, compartmental transmission model (EPICYST) was developed to capture the dynamics of the taeniasis/cysticercosis disease system in the human and pig hosts. Cysticercosis prevalence in humans, an outcome of high epidemiological and clinical importance, was explicitly modelled. A next generation matrix approach was used to derive an expression for the basic reproduction number, R 0. A full sensitivity analysis was performed using a methodology based on Latin-hypercube sampling partial rank correlation coefficient index. Results EPICYST outputs indicate that chemotherapeutic intervention targeted at humans or pigs would be highly effective at reducing taeniasis and cysticercosis prevalence when applied singly, with annual chemotherapy of humans and pigs resulting, respectively, in 94 and 74% of human cysticercosis cases averted. Improved sanitation, meat inspection and animal husbandry are less effective but are still able to reduce prevalence singly or in combination. The value of R 0 for taeniasis was estimated at 1.4 (95% Credible Interval: 0.5–3.6). Conclusions Human- and pig-targeted drug-focussed interventions appear to be the most efficacious approach from the options currently available. The model presented is a forward step towards developing an informed control and elimination strategy for cysticercosis. Together with its validation against field data, EPICYST will be a valuable tool to help reach the WHO goals and to conduct economic evaluations of interventions in varying epidemiological settings

Solar-like oscillations in red giants observed with Kepler: comparison of global oscillation parameters from different methods

The large number of stars for which uninterrupted high-precision photometric timeseries data are being collected with \textit{Kepler} and CoRoT initiated the development of automated methods to analyse the stochastically excited oscillations in main-sequence, subgiant and red-giant stars. Aims: We investigate the differences in results for global oscillation parameters of G and K red-giant stars due to different methods and definitions. We also investigate uncertainties originating from the stochastic nature of the oscillations. Methods: For this investigation we use Kepler data obtained during the first four months of operation. These data have been analysed by different groups using already published methods and the results are compared. We also performed simulations to investigate the uncertainty on the resulting parameters due to different realizations of the stochastic signal. Results: We obtain results for the frequency of maximum oscillation power (nu_max) and the mean large separation () from different methods for over one thousand red-giant stars. The results for these parameters agree within a few percent and seem therefore robust to the different analysis methods and definitions used here. The uncertainties for nu_max and due to differences in realization noise are not negligible and should be taken into account when using these results for stellar modelling.Comment: 11 pages, 9 Figures and 7 tables, accepted for publication in Astronomy and Astrophysic

Three-dimensional Structure of L-2-Haloacid Dehalogenase from Xanthobacter autotrophicus GJ10 Complexed with the Substrate-analogue Formate

The L-2-haloacid dehalogenase from the 1,2-dichloroethane degrading bacterium Xanthobacter autotrophicus GJ10 catalyzes the hydrolytic dehalogenation of small L-2-haloalkanoic acids to yield the corresponding D-2-hydroxyalkanoic acids. Its crystal structure was solved by the method of multiple isomorphous replacement with incorporation of anomalous scattering information and solvent flattening, and was refined at 1.95-Å resolution to an R factor of 21.3%. The three-dimensional structure is similar to that of the homologous L-2-haloacid dehalogenase from Pseudomonas sp. YL (1), but the X. autotrophicus enzyme has an extra dimerization domain, an active site cavity that is completely shielded from the solvent, and a different orientation of several catalytically important amino acid residues. Moreover, under the conditions used, a formate ion is bound in the active site. The position of this substrate-analogue provides valuable information on the reaction mechanism and explains the limited substrate specificity of the Xanthobacter L-2-haloacid dehalogenase.

Old puzzle, new insights: a lithium rich giant quietly burning helium in its core

About 1% of giant stars have been shown to have large surface Li abundances, which is unexpected according to standard stellar evolution models. Several scenarios for lithium production have been proposed, but it is still unclear why these Li-rich giants exist. A missing piece in this puzzle is the knowledge of the exact stage of evolution of these stars. Using low-and-high-resolution spectroscopic observations, we have undertaken a survey of lithium-rich giants in the Kepler field. In this letter, we report the finding of the first confirmed Li-rich core-helium-burning giant, as revealed by asteroseismic analysis. The evolutionary timescales constrained by its mass suggest that Li-production most likely took place through non-canonical mixing at the RGB-tip, possibly during the helium flash.Comment: 16 pages, 4 figures, 1 table, accepted in ApJ Letter