Chemically selective displacers for high-resolution protein separations in ion-exchange systems: effect of displacer-protein interactions

Displacer lead compounds were selected from a commercially available database to identify potential selective displacers for a binary protein mixture in ion exchange chromatography. Parallel batch screening experiments were carried out with these lead compounds to study the effect of displacer concentration on the relative amounts of the proteins displaced. Experiments were conducted with a mixture containing ribonuclease A and alpha-chymo-trypsinogen A which exhibited very similar retention behavior under linear gradient conditions. The batch displacement results indicated that most of these lead compounds were indeed selective for displacing ribonuclease A. In fact, one of these displacers exhibited extremely high selectivity, displacing essentially all of the ribonuclease A while displacing minimal alpha-chymotrypsinogen A at a displacer concentration of 10 mM. These results were validated under column conditions, with the ribonuclease A being displaced and the alpha-chymotrypsinogen A remaining on the column after the displacer breakthrough. In order to examine whether this was mass action or chemically selective displacement, an affinity ranking plot based on the Steric Mass Action (SMA) model was generated, and the results confirmed that this was not a mass action displacement. In order to test the hypothesis that displacer protein binding was playing a role in these separations, Surface Plasmon Resonance (SPR) was carried out. The results suggest that while the chemically selective displacer interacted with alpha-chymo-trypsinogen A, it had no interaction with ribonuclease A. The ability to exploit protein displacer binding in concert with appropriate displacer resin affinities opens up new possibilities for creating selective displacement systems.     

Varied active-site constraints in the klenow fragment of E. coli DNA polymerase I and the lesion-bypass Dbh DNA polymerase

We report on comparative pre-steady-state kinetic analyses of exonuclease-deficient Escherichia coli DNA polymerase I (Klenow fragment, KF-) and the archaeal Y-family DinB homologue (Dbh) of Sulfolobus solfataricus. We used size-augmented sugar-modified thymidine-5'-triphosphate (T(R)TP) analogues to test the effects of steric constraints in the active sites of the polymerases. These nucleotides serve as models for study of DNA polymerases exhibiting both relatively high and low intrinsic selectivity. Substitution of a hydrogen atom at the 4'-position in the nucleotide analogue by a methyl group reduces the maximum rate of nucleotide incorporation by about 40-fold for KF- and about twelve fold for Dbh. Increasing the size to an ethyl group leads to a further twofold reduction in the rates of incorporation for both enzymes. Interestingly, the affinity of KF- for the modified nucleotides is only marginally affected, which would indicate no discrimination during the binding step. Dbh even has a higher affinity for the modified analogues than it does for the natural substrate. Misincorporation of either TTP or T(Me)TP opposite a G template causes a drastic decline in incorporation rates for both enzymes. At the same time, the binding affinities of KF- for these nucleotides drop by about 16- and fourfold, respectively, whereas Dbh shows only a twofold reduction. Available structural data for ternary complexes of relevant DNA polymerases indicate that both enzymes make close contacts with the sugar moiety of the dNTP. Thus, the varied proficiencies of the two enzymes in processing the size-augmented probes indicate varied flexibility of the enzymes' active sites and support the notion of active site tightness being a criterion for DNA polymerase selectivity.     

A universal approach to solvation modeling

Continuum mean-field models that have been carefully designed to address the various electrostatic and nonelectrostatic interactions that develop between a molecule and a surrounding medium are particularly efficient tools for studying the effects of condensed phases on molecular structure, energetics, properties, spectra, interaction potentials, and dynamics. The SM8 model may be combined with density functional theory or Hartree-Fock theory to describe a solute's electronic structure and its self-consistent-field polarization by a solvent. A key feature is the use of class IV charge models to obtain accurate charge distributions (either in the vapor phase or in solution), even when using small basis sets that are affordable for large systems. A second key feature is that nonelectrostatic effects due to cavity formation, dispersion interactions, and changes in solvent structure are included in terms of empirical atomic surface tensions that depend on geometry but do not require atom-type assignments by the user. Use of an analytic surface area algorithm provides very stable energy gradients that allow geometry optimization in solution. The SM8 continuum model, the culmination of a series of SMx models (x = 1-8), permits the modeling of such diverse media as aqueous and organic solvents, soils, lipid bilayers, and air-water interfaces. In addition to predicting accurate transfer free energies between gaseous and condensed phases or between two different condensed phases, SMx models have been useful for predicting the significant influence of condensed phases on processes associated with a change in molecular charge, including acid/base equilibria and oxidation/reduction processes. In this Account, we provide an overview of the algorithms associated with the computation of free energies of solvation in the SM8 model. We also compare the accuracies of the SM8 model with those of other continuum solvation models. Finally, we highlight applications of the SM8 models to compute ionic solvation free energies, oxidation and reduction potentials, and pK(a) values.     

Rhizosphere characteristics of the arsenic hyperaccumulator Pteris vittata L. and monitoring of phytoremoval efficiency

Recently discovered As-hyperaccumulator ferns hold promise for phytoremediation of As-polluted soils. We investigated changes in the rhizosphere characteristics of Pteris vittata (Chinese Brake fern) relevant for its use in phytoextraction. Plants were grown in rhizoboxes filled with soil containing 2270 mg kg(-1) As. Dissolved organic carbon (DOC) concentrations in rhizosphere soil solution were increased by 86% and appeared to enhance total Fe solubility due to complexation reactions. Despite substantial removal of As by the fern, As was not significantly decreased in the rhizsophere soil solution after one cropping, apparently due to the large buffer capacity of the soil and possibly because of ion competition with DOC. However, the difference between 0.05 M (NH4)2SO4-extractable labile As in bulk and rhizosphere soil accounted for 8.9% of total As accumulated in the fern, indicating that As was mainly acquired from less available pools. Moreover, As depletion in the rhizosphere and limited resupply from less available pools were indicated by a 19.3% decreased As flux, measured using the technique of diffusive gradients in thin films (DGT). Modeling of the DGT-soil system was able to show that the rate of release from solid phase to solution in the rhizosphere was one-third of that in the bulk soil. Applying the remedial strategy of bioavailable contaminant stripping, which aims at diminishing the phytoavailable pollutant fraction, DGT can be used as a monitoring tool to evaluate the efficiency of phytoextraction and to study the potential resupply of bioavailable pools after phytoextraction has ceased.     

Lubricating Medical Devices with Fullerene-Like Nanoparticles

In the present work, MoS₂ nanoparticles with fullerene-like structure, and most particularly those doped with minute amounts of rhenium atoms, are used as additive to medical gels in order to facilitate their entry into constricted openings of soft material rings. This procedure is used to mimic the entry of endoscopes to constricted openings of the human body, like urethra, etc. It is shown that the Re-doped nanoparticles reduce the traction force used to retrieve the metallic lead of the endoscope from the soft ring by a factor close to three times with respect to the original gel. The mechanism of the mitigation of both friction and adhesion forces in these systems by the nanoparticles is discussed.     

Dynamics of gas-phase trichloramine (NCl3) in chlorinated, indoor swimming pool facilities

Trichloramine (NCl(3)) is recognized as an irritant of the human respiratory system and other tissues. Processes that lead to volatilization from the liquid phase allow for human exposure to gas-phase NCl(3) in swimming pool settings. The dynamics of these processes are not well defined. A N,N-diethyl-p-phenylenediamine/potassium iodide (DPD/KI)-based wet-chemistry method for measuring gas-phase NCl(3) concentration was verified and applied in chlorinated, indoor swimming pool facilities. Other gas-phase oxidants in the air of indoor pools provided interference of 15% or less. The DPD/KI method was applied for the measurement of gas-phase NCl(3) in four chlorinated, indoor swimming pool facilities. All results showed a correlation between bather loading and gas-phase NCl(3) concentration. The nature of swimmer activities also influenced air quality, presumably because of the effects of these activities on mixing near the gas-liquid interface. PRACTICAL IMPLICATIONS: The activities of swimmers promote transfer of volatile compounds from water to the surrounding air. For chlorinated, indoor pool facilities, this can lead to exposure to gas-phase chemicals that can cause irritation of the respiratory system and other tissues. The focus of this study was on NCl(3), a common disinfection by-product (DBP) in chlorinated pools. However, the conditions that promote NCl(3) transfer are likely to promote transfer of other volatile chemicals from water to air. As such, it is possible that other DBPs formed in pools may also contribute to diminished air quality.     

Temporal trends (1987-2002) of persistent, bioaccumulative and toxic (PBT) chemicals in beluga whales (Delphinapterus leucas) from the St. Lawrence Estuary, Canada

Temporal trends of persistent, bioaccumulative and toxic (PBT) chemicals were examined in beluga whales (Delphinapterus leucas) from the St. Lawrence Estuary (SLE), Canada. Blubber samples of 86 adult belugas were collected from animals stranded on the shore of the SLE between 1987 and 2002 and analyzed for several regulated PBTs, including polychlorinated biphenyls (PCBs), p,p'dichlorodiphenyltrichloroethane (DDT) and its metabolites, chlordane (CHL) and related compounds, hexachlorocyclohexane (HCH) isomers, hexachlorobenzene (HCB) and Mirex. In addition, time trends of tris(4-chlorophenyl)methane (TCPMe) and tris(4-chlorophenyl)methanol (TCPMOH), two compounds that may origin from DDT formulations, were also examined. Concentrations of most of the PBTs examined had exponentially decreased by at least a factor of two (half-life time (t(1/2))<15 years) in beluga between 1987 and 2002 while no increasing trends were observed for any of the PBTs measured. The decreasing trends of PBT concentrations in SLE beluga may be due to a decline in contamination of its diet following North American and international regulations on the use and production of these compounds or by a change in its diet itself or by a combination of both. Some PBTs did not exhibit any significant trends in beluga possibly because the most intense elimination phase subsequent to legislative regulations occurred prior to the 1987-2002 time period. Other chemicals, such gamma-HCH, did not significantly decrease likely because they are still currently used in some restricted applications. Conversely, alpha-HCH showed a significant decreasing trend indicating that SigmaHCHs is not representative of all HCHs. Both TCPMe and TCPMOH exhibited no trends in beluga during the time period examined. The metabolic capacity of SLE beluga has apparently accelerated the depletion of at least one PBT, namely CB-28/31. A significant relationship between the half-life of PBTs in beluga and log Kow was observed for most of the chemicals examined. Several factors are expected to have influenced the temporal changes of PBT concentrations in beluga which limit the usefulness of this species as a bioindicator of changes in PBT contamination in the SLE ecosystem.     

Stable isotope dilution analysis of the Fusarium mycotoxins deoxynivalenol and 3-acetyldeoxynivalenol

Trichothecenes are secondary metabolites produced by several fungi of the Fusarium genus during their growth period. They inhibit protein biosynthesis in eukaryotic cells resulting in numerous toxic effects such as diarrhea, vomiting, and gastro-intestinal inflammation. Considering its occurrence in food and feedstuff, deoxynivalenol (DON) is one of the most important trichothecenes. We report the synthesis of stable isotope labeled 15-d(1)-deoxynivalenol (15-d(1)-DON) from its natural precursor 3-acetyldeoxynivalenol (3-AcDON) as starting material. Furthermore, a method for the analysis of DON and 3-AcDON using HPLC-MS/MS with stable isotope labeled 15-d(1)-DON and 3-d(3)-AcDON as internal standards has been developed. In total, 18 cereal product samples were analyzed with contamination levels ranging from 10-301 microg/kg for DON and 5-14 microg/kg for 3-AcDON. This is the first report of an isotope dilution MS method for the analysis of type B-trichothecenes.     

Effects of a Short Physical Exercise Intervention on Patients with Multiple Sclerosis (MS)

Background: The aim of this prospective randomized controlled trial was to investigate if a short-term endurance or combined endurance/resistance exercise program was sufficient to improve aerobic capacity and maximum force in adult patients (18–65 years) with multiple sclerosis (MS). Methods: All patients performed a three-month exercise program consisting of two training sessions per week, lasting 40 min each, with moderate intensity. All patients had a maximum value of 6 (low to moderate disability) on the Expanded Disability Status Scale (EDSS). One group (combined workout group (CWG); 15 females, 4 males) completed a combined endurance/resistance workout (20 min on a bicycle ergometer, followed by 20 min of resistance training), while the other group (endurance workout group (EWG); 13 females, 5 males) completed a 40 min endurance training program. Aerobic capacity was assessed as peak oxygen uptake, ventilatory anaerobic threshold, and workload expressed as Watts. Maximum force of knee and shoulder extensors and flexors was measured using isokinetic testing. Quality of life was assessed with the SF-36 questionnaire, and fatigue was measured using the Modified Fatigue Impact Scale. Results: Both training groups increased in aerobic capacity and maximum force. EWG, as well as CWG, showed improvement in several subscales of the SF-36 questionnaire and decrease of their fatigue. Conclusion: A short exercise intervention increased both aerobic capacity and maximum force independent of whether endurance or combined endurance/resistance workouts were performed.     

Energy conservation measures in buildings heated by district heating – A local energy system perspective

The extensive energy use in the European building sector creates opportunities for implementing energy conservation measures (ECMs) in residential buildings. If ECM are implemented in buildings that are connected to a district heating (DH) system, the operation of DH plants may be affected, which in turn may change both revenue and electricity production in cogeneration plants. In this study a local energy system, containing a DH supplier and its customer, has been analysed when implementing three ECMs: heat load control, attic insulation and electricity savings. This study is unique since it analyses economic and CO₂ impacts of the ECMs in both a user and a supplier perspective in combination with a deregulated European electricity market. Results show that for the local energy system electricity savings should be prioritised over a reduction in DH use, both from an economic and a global CO₂ perspective. For the DH supplier attic insulation demonstrates unprofitable results, even though this measure affects the expensive peak load boilers most. Heat load control is however financially beneficial for both the DH supplier and the residences. Furthermore, the relation between the fixed and variable DH costs is highlighted as a key factor for the profitability of the ECMs.