The size of the anisotropic domains in a lyotropic liquid crystal is estimated using a new protocol for diffusion NMR. Echo attenuation decays are recorded for different durations of the displacement-encoding gradient pulses, while keeping the effective diffusion time and the range of the wave vectors constant. Deviations between the sets of data appear if there are non-Gaussian diffusion processes occurring on the time-scale defined by the gradient pulse duration and the length-scale defined by the wave vector. The homogeneous length-scale is defined as the minimum length-scale for which the diffusion appears to be Gaussian. Simulations are performed to show that spatial variation of the director orientation in an otherwise homogeneous system is sufficient to induce non-Gaussian diffusion. The method is demonstrated by numerical solutions of the Bloch-Torrey equation and experiments on a range of lamellar liquid crystals with different domain sizes.

The activity and adsorption of three variants of human carbonic anhydrase (HCA) with similar topology but variation in charge and stability were studied in the presence of carboxyl-modified polystyrene nanoparticles of different sizes ranging from 25 nm to 114 nm. The balance of forces driving the adsorption of carbonic anhydrase variants is affected by the physicochemical properties of the protein and the nanoparticle size. All enzymes are totally inhibited upon adsorption due to the transition towards a molten globule like state that lacks enzymatic activity. The size of the particle affects the adsorption of human carbonic anhydrase I and N-terminal truncated human carbonic anhydrase II. Investigations on pH effects indicate that the size of the particle modulates the lateral interactions at the protein layer for these particular variants whose adsorption is mainly driven by electrostatic forces. A third variant, human carbonic anhydrase II, instead shows no strong influence of nanoparticle size which supports an adsorption process mainly driven by the hydrophobic effect.

The aggregation of amyloid β peptides (Aβ) into amyloid fibrils is implicated in the pathology of Alzheimer's disease. In light of the increasing number of proteins reported to retard Aβ fibril formation, we investigated the influence of small hydrophilic model proteins of different charge on Aβ aggregation kinetics and their interaction with Aβ. We followed the amyloid fibril formation of Aβ40 and Aβ42 using thioflavin T fluorescence in the presence of six charge variants of calbindin D9k and single-chain monellin. The formation of fibrils was verified with transmission electron microscopy. We observe retardation of the aggregation process from proteins with net charge +8, +2, -2, and -4, whereas no effect is observed for proteins with net charge of -6 and -8. The single-chain monellin mutant with the highest net charge, scMN+8, has the largest retarding effect on the amyloid fibril formation process, which is noticeably delayed at as low as a 0.01:1 scMN+8 to Aβ40 molar ratio. scMN+8 is also the mutant with the fastest association to Aβ40 as detected by surface plasmon resonance, although all retarding variants of calbindin D9k and single-chain monellin bind to Aβ40.

The fibril formation of the neurodegenerative peptide amyloid β (Aβ42) is sensitive to solution conditions, and several proteins and peptides have been found to retard the process. Aβ42 fibril formation was followed with ThT fluorescence in the presence of polyamino acids (poly-glutamic acid, poly-lysine, and poly-threonine) and other polymers (poly(acrylic acid), poly(ethylenimine), and poly(diallyldimethylammonium chloride). An accelerating effect on the Aβ42 aggregation process is observed from all positively charged polymers, while no effect is seen from the negative or neutral polymers. The accelerating effect is dependent on the concentration of positive polymer in a highly reproducible manner. Acceleration is observed from a 1:500 polymer to Aβ42 weight ratio and up. Polyamino acids and the other polymers exert quantitatively the same effect at the same concentrations based on weight. Fibrils are formed in all cases as verified by transmission electron microscopy. The concentrations of polymers required for acceleration are too low to affect the Aβ42 aggregation process through increased ionic strength or molecular crowding effects. Instead, the acceleration seems to arise from the locally increased Aβ42 concentration near the polymers, which favors association and affects the electrostatic environment of the peptide.

The enzymatic activity of human carbonic anhydrase II (HCAII) was studied in the presence of nanoparticles of different nature and charge. Negatively charged nanoparticles inhibit HCAII whereas no effect is seen for positively charged particles. The kinetic effects were correlated with the strength of binding of the enzyme to the particle surface as measured by ITC and adsorption assays. Moreover, conformational changes upon adsorption were observed by circular dichroism. The main initial driving force for the adsorption of HCAII to nanoparticles is of electrostatic nature whereas the hydrophobic effect is not strong enough to drive the initial binding. This is corroborated by the fact that HCAII do not adsorb on positively charged hydrophobic polystyrene nanoparticles. Furthermore, the dehydration of the particle and protein surface seems to play an important role in the inactivation of HCAII by carboxyl-modified polystyrene nanoparticles. On the other hand, the inactivation by unmodified polystyrene nanoparticles is mainly driven by intramolecular interactions established between the protein and the nanoparticle surface upon conformational changes in the protein.

We developed a chemical analytical procedure for sampling, extracting and determining epidermal skin cortisol concentrations (SCCs) in the harbour porpoise (Phocoena phocoena) using gas chromatography–tandem mass spectrometry. In brief, this involved a pressurized liquid extraction with a two-step solid-phase clean-up. A derivatization step was conducted prior to detection. To evaluate the new assay, cortisol was analysed in three different sample types obtained from four harbour porpoises: skin plates, dorsal fin skin plugs (with and without lidocaine) and epidermal scrapes. Skin cortisol concentrations could be measured using the new assay in the majority of the tested skin samples down to a minimal sample size of 49 mg dry weight (dw). Water content ranged from 10 to 46% in the plug samples, which had SCCs from 2.1 to 77.7 ng/g dw. Epidermal scrape samples had the highest water content (83–87%) and lower SCCs (0.6–15 ng/g dw), while the skin plates had intermediate water contents (60–66%) and SCCs of 2.6–13.0 ng/g dw. SCC was slightly higher in plugs with lidocaine than without (average values of 41 and 33 ng/g dw, respectively). Substantial within-individual variations in cortisol concentrations are also common in other matrices such as blood and hair. Some important factors behind this variation could be e.g. the animal's sex, age, body condition, reproductive stage, and the body region sampled, as well as season, moulting cycles and water temperature. Clearly, more research into SCCs is required. The findings described here represent the first critical steps towards using epidermal skin cell samples to assess chronic stress levels in cetaceans and the development of a widely applicable health-assessment tool in these species.

A kinetic study was carried out on various solvolytic reactions in water/NH4OT/isooctane microemulsions. The NH4OT surfactant is a derivative of the sodium salt of bis(2-ethylhexyl) sulfosuccinate (NaOT or AOT), where the Na+ counterion has been replaced by NH4+. The counterion substitution effects the phase diagram of the system, and therefore, NH4OT-based microemulsions with high water content reaching values of W = 350 (W = [H2O]/[NH4OT]) can be obtained. The presence of high W values suggests a transition in the microemulsion microstructure from water-in-oil (w/o) to oil-in-water (o/w), as was confirmed by conductivity and H-1 NMR self-diffusion measurements. The interpretation of the kinetic studies in terms of pseudophase formalism allows us to analyze the effect of the microemulsion on chemical reactivity, regardless of its microstructure. It has been confirmed that the values of the solvolytic rate constants at the interphase of oil-in-water microemulsions are similar to those obtained for aqueous SDS systems, showing that the hydration degree of the interphase of the oil-in-water microemulsions is independent of W. The influence of the surfactant counterion on the solvolytic rate constants was analyzed by comparing HOT-, NaOT-, and NH4OT-based microemulsions. An important influence on the rate constants caused by the changes in the structural properties of water has been observed as was confirmed by the water H-1 NMR signals.

The chemical behavior of beta-cyclodextrin/nonionic surfactant mixed systems has been investigated using the basic hydrolysis of N-methyl-N-nitroso-p-toluenesulfonamide as a chemical probe. The experimental results prove that at the cmc, there are significant quantities of uncomplexed beta-CD in equilibrium with the micellar aggregates. In contrast to the expected situation, the percentage of uncomplexed beta-CD in equilibrium with the micellar system increases on increasing the hydrophobicity of the surfactant molecule. This behavior is due to the existence of two simultaneous processes: complexation of surfactant monomers by cyclodextrin and the process of self-assembly to form micellar aggregates. The autoaggregation of surfactant monomers is expected to be more important than the complexation process in this mixed system. Varying the hydrophobicity of the surfactant monomer enabled us to determine that the percentages of uncomplexed cyclodextrin in equilibrium with the micellar system were in the range of 5-95%.

A systematic study on the solvolysis reaction of substituted benzoyl chlorides in the presence of zwitterionic vesicles of dipalmitoyl phosphatidylcholine (DPPC) has been performed. Size, shape, surface charge, and polarity of the interface of the vesicular aggregates were determined using various techniques. The application of the pseudophase formalism allowed us to obtain the thermodynamic and kinetic coefficients characteristic of the reaction. The effects of vesicular aggregates on the solvolysis of benzoyl chlorides, which are known to be sensitive to the physical properties of the medium, depend on the nature of the substrate. For benzoyl chlorides with electron-donating groups, which react predominantly through a dissociative mechanism which is strongly affected by medium properties, the rate constant decreases as the vesicle concentration increases. On the other hand, for benzoyl chlorides with electron-withdrawing groups, which react mainly via an associative pathway, DPPC vesicles catalyze the solvolysis reaction.

The solvolysis reactivity of benzoyl chlorides entails a high sensitivity on medium properties. A systematic study of the reaction of a series of these substrates, varying the electron-withdrawing character of the substituent, has been performed in nonionic microemulsions. The kinetic effects due to variation of microemulsion compositions can be assigned to modifications in system properties, to be precise, to modifications in interface properties. Microemulsion properties that are obtained from kinetic analysis of solvolysis show a good agreement with the characterization of the microemulsion that was made via H-1 NMR and solvatochromic fluorescence probes. Benzoyl chlorides with electron-donating groups react through a dissociative mechanism, whereas electron-withdrawing groups favor an associative mechanism. A comparative analysis of reactivity between the different substrates at the interface shows a variation in the contributions of both reaction pathways, associative and dissociative, to the whole reaction mechanism. The confined media shift the point where the mechanism changes from an associative to a dissociative pathway, far away from the turning point in water. Furthermore, the change in mechanism can be modulated by modification of the microemulsion composition.

The complexation between Ni2+ and pyridine-2-azo-p-dimethylaniline (PADA) was studied in isooctane/polyoxyethylenglycol dodecyl ether (Brij30)/water microemulsions at 25 degrees C. The apparent complexation constant depends on microemulsion composition. The proposed model, which takes into account the heterogeneity of the system at the microscopic scale, allows us to determine the distribution constants of both reactants and the complexation constant at the interface. The complex is less stable in microemulsion than in water due to a more efficient hydration of the nickel hexahydrate coordination complex that arises from the interaction between the polar head group of the surfactant and the interfacial water. (c) 2006 Elsevier B.V. All rights reserved.

A study was carried out on the acid denitrosation of N-methyl-N-nitroso-p-toluenesulfonamide (MNTS) in mixed systems made up of linear (geminal and terminal) alkyl diols and beta-cyclodextrin (CD). The alkyl diols used allowed us to vary the length of the hydrocarbon chain from 2 to 6 carbon atoms. The observed rate constant, k(obs), decreases in the presence of CD. The inhibition profile decreases as the as the number of carbons in the chain is increased. This behaviour can be interpreted as a consequence of a balance between the complexation processes of MNTS and the alkyl diols by the CD. At a constant CD concentration and increase in the diols concentration decreases the concentration of free cyclodextrin available to complex with MNTS molecules and therefore produces an increases in the observed rate constant. The results were interpreted in terms of two different models; trough the presupposition and non-presupposition of a stoichiometry for the CD-diols complex. Both models agreed quite well and allow us to determine the uncomplexed cyclodextrin concentration in each case as well as the stoichiometry of the complexes. The binding constant for both types of alkane diols increase with increasing the number of carbon in the chain. Besides, the binding constant of the alpha,beta-alkane diols is higher than for the analog alpha,beta-alkane diols. One of the main consequences of this study is that the acid denitrosation of MNTS can be use to obtain the stochiometry of the CD-diol complexes and to monitor the free cyclodextrin concentration.

In this contribution the influence of beta-cyclodextrin (CD) on the behavior of aqueous systems containing vesicles of dipalmitoyl phosphatidyl choline (DPPC) has been studied by determining the kinetics of the solvolysis reaction of substituted benzoyl chlorides whose solvolysis reactivity entails a high sensitivity on media properties. The application of the pseudophase formalism allowed us to obtain the thermodynamic and kinetic coefficients characteristic of the reaction, which are essentially independent of the concentration of CD. We were able to determine the percentages of uncomplexed cyclodextrin in equilibrium with the vesicular system which were in all cases compatible with 100%. The obtained results led us to conclude that the properties of DPPC vesicles are not affected by the presence of CD in the medium and there is no type of interaction between the CD and the vesicular surfactant monomers and, therefore, all cyclodextrin is present in the mixed system as uncomplexed cyclodextrin.

Human carbonic anhydrases (hCAs) belong to a well characterized group of metalloenzymes that catalyze the conversion of carbonic dioxide into bicarbonate. There are currently 15 known human isoforms of carbonic anhydrase with different functions and distribution in the body. This links to the relevance of hCA variants to several diseases such as glaucoma, epilepsy, mountain sickness, ulcers, osteoporosis, obesity and cancer. This review will focus on two of the human isoforms, hCA I and hCA II. Both are cytosolic enzymes with similar topology and 60% sequence homology but different catalytic efficiency and stability. Proteins in general adsorb on surfaces and this is also the case for hCA I and hCA II. The adsorption process can lead to alteration of the original function of the protein. However, if the function is preserved interesting biotechnological applications can be developed. This review will cover the knowledge about the interaction between hCAs and nanomaterials. We will highlight how the interaction may lead to conformational changes that render the enzyme inactive. Moreover, the importance of different factors on the final effect on hCAs, such as protein stability, protein hydrophobic or charged patches and chemistry of the nanoparticle surface will be discussed.

The fibrillation process of the islet amyloid polypeptide (IAPP) and its fragment (IAPP(20-29)) was studied by means of Thioflavin T (ThT) fluorescence and transmission electron microscopy in the absence and presence of N-isopropylacrylamide:N-tert-butylacrylamide (NiPAM:BAM) copolymeric nanoparticles. The process was found to be strongly affected by the presence of the nanoparticles, which retard protein fibrillation as a function of the chemical surface properties of the nanoparticles. The NiPAM:BAM ratio was varied from 50:50 to 100:0. The nanoparticles with higher fraction of NiPAM imposed the strongest retardation of IAPP and IAPP(20-29) fibrillation. These particles have the strongest hydrogen bonding capacity due to the less bulky N-isopropyl group and thus less steric hindrance of the hydrogen-bonding groups of the nanoparticle polymer backbone. Kinetic fibrillation data, as monitored by ThT fluorescence and supported by surface plasmon resonance experiments, suggest that the peptide is strongly absorbed onto the surface of the nanoparticles. This interaction reduces the concentration of peptide free in solution available to proceed to fibrillation which results in an increased lag time of fibrillation, observed as a delayed onset of ThT fluorescence increase, plus a reduction of the amount of fibrils formed as indicated by the equilibrium values at the end of the fibrillation reaction. For the fragment (IAPP(20-29)), the presence of nanoparticles changes the mechanism of association from monomers to fibrils, by interfering with early oligomeric species along the fibrillation pathway.

Diffusion measurements by nuclear magnetic resonance (NMR) spectroscopy were used to investigate the host-guest association between beta-cyclodextrin (CD) and alkyltrimethylammonium bromide surfactants with different chain lengths, ranging from 6 up to 16 carbons. The scope and limitations of the method in the study of formation of inclusion complexes are discussed. The influences of the presence of CD in the micellization process have been studied, and the apparent critical micellar concentration and the self-diffusion coefficients of the species present in the systems have been calculated. The stoichiometries of the different complexes have been determined. Evidence for the formation of a 2:1 complex in the case Of C(16)TAB has been found.

Surfactants form association complexes with cyclodextrins. In the present investigation we have used NMR-diffusometry and electrical conductivity to follow the interactions which take place between ss-cyclodextrm and a bolaform surfactant: dodecane 1, 1 2-bis(trimethylammonium bromide). Both H-1 NMR self-diffusion and conductometry data indicate the formation of a 1: 1 inclusion complex. Assuming this stoichiometry, it was possible to calculate the association constant; from the analysis of the self-diffusion coefficients of free ss-cyclodextrin and the bolaform surfactant an association constant K = 3 x 10(3) Mb(-1) was obtained while the analysis of conductivity data gave a comparable value of K = 2.5 x 10(3) M-1. (c) 2006 Elsevier Inc. All rights reserved.

The fibrillation kinetics of the amyloid β peptide is analyzed in presence of cationic polystyrene nanoparticles of different size. The results highlight the importance of the ratio between the peptide and particle concentration. Depending on the specific ratio, the kinetic effects vary from acceleration of the fibrillation process by reducing the lag phase at low particle surface area in solution to inhibition of the fibrillation process at high particle surface area. The kinetic behavior can be explained if we assume a balance between two different pathways: first fibrillation of free monomer in solution and second nucleation and fibrillation promoted at the particle surface. The overall rate of fibrillation will depend on the interplay between these two pathways, and the predominance of one mechanism over the other will be determined by the relative equilibrium and rate constants.

Copolymeric NiPAM:BAM nanoparticles of varying hydrophobicity were found to retard fibrillation of the Alzheimer's disease-associated amyloid beta protein (Abeta). We found that these nanoparticles affect mainly the nucleation step of Abeta fibrillation. The elongation step is largely unaffected by the particles, and once the Abeta is nucleated, the fibrillation process occurs with the same rate as in the absence of nanoparticles. The extension of the lag phase for fibrillation of Abeta is strongly dependent on both the amount and surface character of the nanoparticles. Surface plasmon resonance studies show that Abeta binds to the nanoparticles and provide rate and equilibrium constants for the interaction. Numerical analysis of the kinetic data for fibrillation suggests that binding of monomeric Abeta and prefibrillar oligomers to the nanoparticles prevents fibrillation. Moreover, we find that fibrillation of Abeta initiated in the absence of nanoparticles can be reversed by addition of nanoparticles up to a particular time point before mature fibrils appear.

Nanoparticles interfere with protein amyloid formation. Catalysis of the process may occur due to increased local protein concentration and nucleation on the nanoparticle surface, whereas tight binding or a large particle/protein surface area may lead to inhibition of protein aggregation. Here we show a clear correlation between the intrinsic protein stability and the nanoparticle effect on the aggregation rate. The results were reached for a series of five mutants of single-chain monellin differing in intrinsic stability toward denaturation, for which a correlation between protein stability and aggregation propensity has been previously documented by Szczepankiewicz et al. [Mol. Biosyst.20107 (2), 521-532]. The aggregation process was monitored by thioflavin T fluorescence in the absence and presence of copolymeric nanoparticles with different hydrophobic characters. For mutants with a high intrinsic stability and low intrinsic aggregation rate, we find that amyloid fibril formation is accelerated by nanoparticles. For mutants with a low intrinsic stability and high intrinsic aggregation rate, we find the opposite--a retardation of amyloid fibril formation by nanoparticles. Moreover, both catalytic and inhibitory effects are most pronounced with the least hydrophobic nanoparticles, which have a larger surface accessibility of hydrogen-bonding groups in the polymer backbone.

Solvolysis of benzoyl halides has been studied in nonionic isooctane/Brij30/water microemulsions. The reaction takes place only at the microemulsion interface due to the low solubility of benzoyl halides. The variation of the properties (water content and hydration water nucleophilic character) of the microemulsion interface as the amount of water in the system decreases favors mechanistic changes in the solvolytic reaction. The true rate and equilibrium constants at the interface were obtained by means of the formalism of the pseudophase. Rate constant values at the interface change as the water content of the microemulsion is modified. The changes are consistent with a shift of mechanism from dissociative toward associative. The predominance of either dissociative or associative mechanism is dependent on the characteristics of the leaving group. The associative character decreases as the salient group of the benzoyl halides improves. Moreover, the associative pathway is strongly favored in the microemulsion system studied.

The solvolysis of substituted benzoyl chlorides is sensitive both to substituent electronic effects and to medium effects. The solvolysis reactions of substituted benzoyl chlorides have been analyzed in the presence of nonionic micelles. The reaction is inhibited or catalyzed depending on the reaction mechanism, dissociative or associative, respectively. The micellar effects observed can be related to the low water content and low polarity of the interface as well as an increase of the nucleophilic character of the interfacial water. Moreover, the effect of the micellar surface charge on the solvolysis mechanism with high associative character was systematically studied. Mixed micelles of nonionic-ionic surfactants with a variable ionic content were prepared and characterized regarding charge and polarity. A correlation between the net charge of the micelles and the rate constants at the micellar interface was observed. The results suggest that the transient state for this mechanism is highly stabilized in a positively charged environment while the negative surface given by anionic micelles strongly inhibit the solvolysis reaction.

Pancreatitis is a common disease in canine and felines and can be diagnosed by measuring the concentration of Canine Pancreas-specific Lipase (cPL) or Feline Pancreas-specific Lipase (fPL) in serum. Based on the concentration of cPL/fPL, the patient is then classified in different diagnostic categories (normal value, gray zone or indication of pancreatitis). Spec cPL and Spec fPL is currently the golden standard method for analysis of cPL and fPL. New methods have been developed for the quantitative measurement of pancreatic lipases. Vcheck V200, being one example, utilizing a fluorescent immunoassay for quantification of the lipase. The aim of this study was to evaluate the cPL and fPL analysis on the Vcheck V200 and to examine if there was a significant difference (p≤0,05) when comparing the result from Vcheck V200 with the results from a reference laboratory. The concentration of cPL (n=37) and fPL (n=29) in serum from canine or felines were analyzed using Vcheck V200. The samples were also sent to the reference laboratory where Spec cPL and Spec cPL were performed. A Bland-Altman plot comparison between the two methods showed a large spread for both analysis of cPL and fPL. Comparison of the specific values for analysis of cPL and fPL between the two methods revealed a significant difference (p<0,05). 27% of the dog samples were categorized differently according to the two methods and this difference was significant (p<0,05). 24% of the cat samples were categorized differently and no significant difference were observed (p=0,257). This study indicates that the difference between the two methods was significant, besides the classification of cat samples. Considering this and the lack of quality assurance regarding analysis of fPL due to lack of controls, the cPL/fPL analysis on Vcheck V200 cannot replace the Spec cPL or Spec fPL at present.

EPR spectroscopy and product isolation have been used to investigate the initiation of spontaneous copolymerizations of donor and acceptor monomers. The results establish the presence of tetramethylene diradicals as the only experimentally supported intermediates in the initiation for these spontaneous copolymerizations. No evidence supporting a Mayo cycloaddition mechanism, electron transfer, or charge-transfer complex initiation was found. The reaction of an olefin activated by three electron-accepting groups, such as dimethyl cyanofumarate, methyl 3,3-dicyanoacrylate, or trimethyl ethylenetricarboxylate, with a styrene having unsubstituted ortho positions was used in this study. In the presence of the spin trap 2-methyl-2-nitrosopropane (t-BuNO), aminoxyls with characteristic EPR spectra of high intensity were detected. Reaction of 4-methoxystyrene and dimethyl cyanofumarate in the presence of 2,2,6,6-tetramethylpiperidine-N-oxyl (TEMPO) led to a diamagnetic 1:1:1 adduct, 4-cyano-6-methoxy-3,4-dimethoxycarbonyl-1-piperidinyloxytetrahydronaphthalene, which was subjected to an X-ray crystallographic study. Treatment of the TEMPO adduct with t-BuNO in chloroform gave a solution containing TEMPO and the same aminoxyl as that formed from 4-methoxystyrene, dimethyl cyanofumarate, and t-BuNO. These aminoxyls were assigned the structure of spin adducts of tetrahydronaphthalen-1-yl radicals, formed by initial trapping of the diradical of the donor and acceptor olefin at the more reactive radical center, followed by cyclization into the aromatic ring. This proposed mechanism provides an alternative to the commonly accepted Mayo initiation mechanism.

Food-emulsions often have high volume fractions of dispersed phase and are thus expected to show coalescence during emulsification, however, food-emulsion coalescence is difficult to measure in homogenizer equipment. This study experimentally estimates the rates of fragmentation and coalescence in a high viscosity and high volume fraction model emulsion subjected to pilot-scale rotor-stator mixing in order to quantify the relative effect of coalescence and discuss the mechanism of coalescence during batch processing of high-fat emulsion foods. Rate constants of both processes are estimated using a previously suggested method relying on parameter fitting from the dynamic evolution of the total number of emulsion drops (Hounslow and Ni, 2004). The results show substantial coalescence taking place. Scaling of rates with respect to rotor tip speed suggests coalescence and fragmentation controlled by a turbulent viscous mechanism. (C) 2015 Elsevier Ltd. All rights reserved.

Vitellogenin (VTG) is a well-known biomarker for endocrine disruption and the measurement of alkali-labile protein bound phosphate (ALP) has been used as an indirect quantification method for VTG in fish, clam and mussel. One limitation of the ALP method has been the insufficient detection limit but with the method presented here this obstacle has been overcome, enabling measurement in fish species with very low background levels and hence a wider usage of ALP measurements in the future. In a previous publication, relying on spectrophotometric measurement with molybdate, we presented an improved protocol for ALP measurement in fish plasma. The sample preparation in that paper has here been combined with an improved spectrophotometric determination using malachite green as colour reagent. The spectrophotometric measurement was optimised with respect to acidity and reagent concentration. The validated method has a detection limit of 0.3?µg? ?ml?1 plasma, which is 10 times lower than previous spectrophotometric methods using molybdate reagent, and an intra-assay variation of 7%. The new method was used for screening of endocrine effects by placing juvenile rainbow trout at three locations along the Vallkärra brook, Lund Sweden, which receives leachate water from a nearby covered landfill. In comparison with reference fish, ALP was significantly higher (66%) at the 99% confidence level in fish placed close to the landfill. ALP was also significantly higher (33%) at the 95% confidence level in fish from the group placed further downstream. Fish placed in a brook branch not receiving leachate were unaffected. The low levels of ALP in reference fish (5.5?±?0.7 SD ?µg/ml plasma) could not have been quantified with such precision with another method for ALP measurement. The indirect quantification of VTG as ALP is a more economic alternative compared to quantification with immunological methods.

Vitellogenin (VTG) is a well known protein biomarker for exposure to environmental estrogens and possible endocrine disruption in fish. VTG is very dominant in plasma after the onset of vitellogenesis and the protein is heavily phosphorylated. This enables indirect quantification through measurement of alkali-labile protein bound phosphate (ALP) as an alternative to the more expensive enzyme linked immunosorbent assay. Good correlation has previously been shown between ALP and actual VTG levels but little effort has been made to investigate the method in an analytical way e.g., to assure the origin of the measured phosphate. During this method development care has been taken to rule out non-VTG sources of phosphate such as phospholipids and free phosphate in the blood plasma. Sample preparation has been simplified and unnecessary steps have been omitted. The common spectrophotometric measurement for ALP involves measurement at two wavelengths and calculation of corrected absorbance values. With a quick phase separation step the spectrophotometric phosphate determination using molybdic acid and ascorbic acid has been improved and all matrix interference has been eliminated. The final ALP method presented here has a detection limit of 3.2 µg PO43-/ml plasma which is six times lower than similar methods and it also has less variability. A high sample throughput in comparison to previous ALP methods is possible after scaling down sample and reagent volumes to fit in a 96 well microtiter plate. The cost for buying all chemicals and plastic consumer goods for setting up the indirect protocol for the analysis of 1000 samples is only circa 350 euro. This is only 1% of the material cost for buying commercially available test kit for direct quantification of VTG in the same number of samples. The ALP method should thus be of interest also for applied scientists outside advanced research laboratories.

When steroid hormones are emitted into the environment, they may have harmful effects on the reproduction system of aquatic life. Until now, research has primarily focused on human excretion, demonstrating that steroid hormones reach the aquatic environment due to insufficient removal in waste water treatment processes. However more recently, it has been revealed that agricultural practices also may add to the environmental burden of steroid hormones. So far, research activities have mainly focused on steroid estrogens, but also androgens, progestagens and glucocorticoids, expressed in the vertebrate steroidogenesis, may occur at substantial levels in animal manure and should be addressed. In agricultural practices the animal manure can be applied to the soil as raw manure, but also as a solid or liquid manure fraction, since current livestock production facilities utilizes a recently developed technology, which separates raw animal manure into a solid and a liquid fraction.This technology offers an improved handling and refined distribution of the manure nutrients to the farmlands and the possibility to reduce the environmental impact of manure nutrients, especially avoiding the surplus load of phosphorous. In the present work we investigated the distribution of 9 steroid hormones (pregnenolone, progesterone, dehydroepiandrosterone, androstenedione, testosterone, dihydrotestosterone, estrone, 17α-estradiol and 17β-estradiol) in raw manure and manure separates from 10 to 15 different pig farms in Denmark utilizing 4 different separation technologies. Furthermore, we investigated a possible relationship between the steroid hormone concentration and the different manure fractions and separation technologies. The chemical steroid hormone analysis was done by inverse and integrated clean-up pressurized liquid extraction, and further cleaned by a two step solid-phase extraction before derivatization and finally analyzed by GC-MS/MS.It was found that the steroid hormones were predominant in the solid manure separate calling for manure management strategies to reduce the content of steroid hormones in separated manure solid fraction. This could potentially be achieved through composting or anaerobic digestion for biogas production of the solid fraction; however, the effects of these technologies on steroid hormones need to be verified.

Newly developed treatment technologies are capable of separating livestock manure into a liquid fraction and a solid fraction using sedimentation, mechanical, and/or chemical methods. These technologies offer a potential means of distributing nutrients to agricultural lands without the unwanted environmental risks associated with the release of steroid hormones to adjacent waterways. To assess the potential benefit of these technologies in reducing the level of release of steroid hormones to adjacent waterways, distribution profiles of nine steroid hormones (pregnenolone, progesterone, dehydroepiandrosterone, androstenedione, testosterone, dihydrotestosterone, estrone, 17α-estradiol, and 17β-estradiol) were determined in raw swine manure, and in solid and liquid fractions separated from ten full-scale manure separation systems. Steroid hormone concentrations, normalized for nitrogen content, were significantly higher in separated solids than in liquids. If separated liquids are applied instead of raw manure, steroid hormone loading can be reduced by a factor of 2 at a constant nitrogen fertilization level.

Semiochemicals are chemicals used to communicate. Animals tend to use these e.g. to locate food sources or to find a suitable mate. In this study, the sex pheromone of the Indian meal moth, Plodia interpunctella, was analysed. Since this is an economically important species, it is mass-reared in labs and science centers worldwide for experimental purposes. A culture of these moths was brought into the lab at Lund University for studies and has after that served as a model species demonstrating up-wind pheromone-mediated male flight in different courses held by the university. As years went by, the culture got less successful in up-wind flights, most probably because of inbreeding and bottleneck effects, and therefore, a new culture was taken in. This study focuses on using various experiments to see if there was a behavioral and/or physiological difference between the two cultures. Results show a significant difference in behavioral traits (frequency of calling behavior in females and in male up-wind flights) but not in physiological traits (female pheromone production or male antennal response). This study discusses some effects of mass-reared lab cultures.

Det här examensarbetet fokuserar på hur man kan kombinera två innehållsaspekter i undervisning med barn i förskolan. Innehållet som bearbetas är naturvetenskap och estetik. Naturvetenskapsdelen i arbetet innefattar kemiska reaktioner och den estetiska delen innefattar drama. Dramat kommer att användas som ett verktyg för att lära om naturvetenskap med fokus på kemiska reaktioner. Studiens insamlingsmetod innefattar filmning och ljudinspelning. Resultatet i studien visar att barnens perspektiv blir synligt genom att barnen får berätta sina tankar om kemiska vätskor. Studiens resultat visar även att ett intresse har väckts bland barnen kring kemiska vätskor och detta lägger en grund för förskollärare att arbeta vidare med och utgå från barnens intresse.

In this chapter, a detailed review of the current understanding of the physical processes of droplet breakup in a high-pressure homogenizer (HPH) is presented, covering breakup mechanisms by laminar shear in the gap inlet and its boundary layers, by local turbulence in the gap exit jet, and by cavitation. Experimental evidence of the effetcts of homogenization pressure, Thoma number, dispersed and continuous phase viscosity, and dispersed phase volume fraction in relation to implications on dominant mechanisms of droplet breakup is also discussed.

We studied the pH-dependence of ribosome catalyzed peptidyl transfer from fMet-tRNA(fMet) to the aa-tRNAs Phe-tRNA(Phe), Ala-tRNA(Ala), Gly-tRNA(Gly), Pro-tRNA(Pro), Asn-tRNA(Asn), and Ile-tRNA(Ile), selected to cover a large range of intrinsic pK(a)-values for the α-amino group of their amino acids. The peptidyl transfer rates were different at pH 7.5 and displayed different pH-dependence, quantified as the pH-value, pK(a)(obs), at which the rate was half maximal. The pK(a)(obs)-values were downshifted relative to the intrinsic pK(a)-value of aa-tRNAs in bulk solution. Gly-tRNA(Gly) had the smallest downshift, while Ile-tRNA(Ile) and Ala-tRNA(Ala) had the largest downshifts. These downshifts correlate strongly with molecular dynamics (MD) estimates of the downshifts in pK(a)-values of these aa-tRNAs upon A-site binding. Our data show the chemistry of peptide bond formation to be rate limiting for peptidyl transfer at pH 7.5 in the Gly and Pro cases and indicate rate limiting chemistry for all six aa-tRNAs.

Nanoparticles present enormous surface areas and are found to enhance the rate of protein fibrillation by decreasing the lag time for nucleation. Protein fibrillation is involved in many human diseases, including Alzheimer's, Creutzfeld-Jacob disease, and dialysis-related amyloidosis. Fibril formation occurs by nucleation-dependent kinetics, wherein formation of a critical nucleus is the key rate-determining step, after which fibrillation proceeds rapidly. We show that nanoparticles (copolymer particles, cerium oxide particles, quantum dots, and carbon nanotubes) enhance the probability of appearance of a critical nucleus for nucleation of protein fibrils from human beta(2)-microglobulin. The observed shorter lag (nucleation) phase depends on the amount and nature of particle surface. There is an exchange of protein between solution and nanoparticle surface, and beta(2)-Microglobulin forms multiple layers on the particle surface, providing a locally increased protein concentration promoting oligomer formation. This and the shortened lag phase suggest a mechanism involving surf ace-assisted nucleation that may increase the risk for toxic cluster and amyloid formation. It also opens the door to new routes for the controlled self-assembly of proteins and peptides into novel nanomaterials.

The effect of the buffer formulation in terms of buffer identity and ionic strength on the interaction between chicken egg lysozyme and carboxyl-modified polystyrene nanoparticles has been systematically studied. The time evolution of the fluorescence of a reporter molecule shows that lysozyme interacts with the nanoparticles in all the studied conditions. The interaction results in changes in protein conformation and decrease of the colloidal stability of nanoparticles. In absence of a background salt the rate of adsorption is affected mainly by the ionic strength of the buffer solution, although, specific buffer effects may contribute to a certain extent. The identity of the different buffer components does not significantly alter the dynamics of the process in presence of salt at constant ionic strength. However, an increase of ionic strength leads to slower processes indicating that the adsorption is affected by the presence of increasing number of ions in solution.

The major aim of our current work is to develop a deep understanding of biological effects of nanoparticles and how these effects are mediated by proteins that are adsorbed on the nanoparticles under different biological circumstances. Due to their small size, nanoparticles have distinct properties compared to the bulk form of the same materials, and these properties are rapidly revolutionizing many areas of medicine and technology. However, relatively little is known about the interaction of nanoscale objects with biological systems, as this requires quite different concepts from more established nanoscience. Thus, we have argued that in a biological fluid, proteins associate with nanoparticles, and it is the amount and presentation of the proteins on the surface rather than the particles themselves that are the cause of numerous biological responses. It is this outer layer of proteins that is seen by the biological cells, and leads to their responses. We are developing novel techniques to identify and quantify the proteins that are consistently associated with nanoparticles, as a function of the nanoparticle size, shape, and surface properties, and to correlate the adsorbed protein identities with their association and dissociation rates to and from the nanoparticles. We also seek to understand the degree of conformational change that they undergo upon adsorption to the nanoparticles. In essence, we wish to create "epitope maps" of the protein corona that surrounds nanoparticles in biological solutions, as it is the particle-protein complex that is the biologically active entity. (c) 2007 Elsevier B.V. All rights reserved.

The efforts spent on judgments of how to perform sampling are often small compared to other

steps in the analytical procedure. As a result decisions may be based on irrelevant results. If

the sampling is intended to provide information how to reduce and control environmental

pollution, it is important to design the sampling strategy according to scientific principles.

Sampling will be discussed in relation to determinations of organic as well as inorganic

species in different types of polluted matrices, .e.g. water, sediments and air. Examples will

be taken from our own research projects about treatment processes for contaminated

matrices.. Accurate sampling involves a basic planning including objectives, cost

effectiveness versus the budget, variability of contamination leading to statistical

considerations, site accessibility and robustness of the used devices. A strategy for the

sampling, often in a form of a scheme, is needed. The Laqua Protocol developed within our

research projects will be presented. This protocol is flexible and dynamic and can be altered

and optimized based on the demands at the individual sampling site. Sampling of complicated

matrices often creates problems with interfering agents and breakdown of unstable analytes.

In this type of samples the concentrations of analytes are generally several orders of

magnitudes lower than the concentrations of major constituents in the sample. Accordingly

the advantages of an early work-up, preferably in connection with a sampling procedure, is

paid especially attention

The adsorption induced conformational changes of human carbonic anhydrase I (HCAi) and pseudo wild type human carbonic anhydrase II truncated at the 17th residue at the N-terminus (trHCAii) were studied in presence of nanoparticles of different sizes and polarities. Isothermal titration calorimetry (ITC) studies showed that the binding to apolar surfaces is affected by the nanoparticle size in combination with the inherent protein stability. 8-Anilino-1-naphthalenesulfonic acid (ANS) fluorescence revealed that HCAs adsorb to both hydrophilic and hydrophobic surfaces, however the dynamics of the unfolding at the nanoparticle surfaces drastically vary with the polarity. The size of the nanoparticles has opposite effects depending on the polarity of the nanoparticle surface. The apolar nanoparticles induce seconds timescale structural rearrangements whereas polar nanoparticles induce hours timescale structural rearrangements on the same charged HCA variant. Here, a simple model is proposed where the difference in the timescales of adsorption is correlated with the energy barriers for initial docking and structural rearrangements which are firmly regulated by the surface polarity. Near-UV circular dichorism (CD) further supports that both protein variants undergo structural rearrangements at the nanoparticle surfaces regardless of being "hard" or "soft". However, the conformational changes induced by the apolar surfaces differ for each HCA isoform and diverge from the previously reported effect of silica nanoparticles.

Mixture effects of 3 model endocrine disruptors, prochloraz, ketoconazole, and genistein, on steroidogenesis were tested in the adrenocortical H295R cell line. Seven key steroid hormones (pregnenolone, progesterone, dehydroepiandrosterone, androstenedione, testosterone, estrone, and 17β-estradiol) were analyzed using gas chromatography and tandem mass spectrometry (GC-MS/MS) to investigate the effects throughout the steroidogenic pathway. Current modeling approaches often rely on models assuming compounds acting independently and that the individual effects in some way can be summarized to predict a mixture effect. In H295R cells with an intact steroidogenic pathway, such assumptions may not be feasible. The purpose of this study was therefore to evaluate whether effects of a mixture with differing modes of action followed or deviated from additivity (concentration addition) and whether the H295R cell line was suitable for evaluating mixture toxicity of endocrine disruptors with different modes of action. The compounds were chosen because they interfere with steroidogenesis in different ways. They all individually decrease the concentrations of the main sex steroids downstream but exert different effects upstream in the steroidogenic pathway. Throughout the study, we observed lowest observed effect concentrations of mixtures at levels 2 to 10 times higher than the predicted EC50, strongly indicating antagonistic effects. The results demonstrate that chemical analysis combined with the H295R cell assay is a useful tool also for studying how mixtures of endocrine disruptors with differing modes of action interfere with the steroidogenic pathway and that existing models like concentration addition are insufficient in such cases. Furthermore, for end points where compounds exert opposite effects, no relevant models are available.

The interaction between beta-cyclodextrin (CD) and gemini surfactant of the type alkyl-alpha,omega-bis(dodecyldimethyl-ammonium bromide) with different spacer lengths of 2, 8, and 10 carbons has been investigated by means of electric conductivity (EC) and proton self-diffusion NMR at 298 K. The formation of a 2: 1 (CD: gemini) complex in a two-step mechanism is observed with the first association constant (K-11) higher than the second one (K-21), but both relatively small in comparison with single C-12-tailed surfactant. The value of the association constants increased with spacer length both for the first and second associated CD, which indicates that the available space on the gemini molecule is important. The magnitudes of the association constant both for the first and second complexation are discussed. The first association constant is small ( when compared with the homologous single-chain surfactant) due to hydrophobic interaction between the hydrocarbon tails within the gemini molecule, while the second association constant shows no cooperativity and its magnitude is discussed in terms of steric constrains.

Poor quality antibiotic medicines in circulation in Sub-Saharan Africa continue to be a burden. Pharmaceutical trade in substandard and counterfeit medicines is on the rise. The chemical quality of antibiotics dispensed in health facilities and recognised drug outlets in Ghana, when compromised, could be a major drawback to efforts made in fighting antibiotic resistance globally. To improve on antibiotic drug quality monitoring, a liquid chromatography-tandem mass spectrometry (LC-MS/MS) methodology, which is capable of quantifying thirteen antibiotics in drug products, was developed and validated in present work. The methodology was applied to various drug products including tablets, capsules, suspensions, syrups, intravenous and injection solutions as well as ear and eye droplets used as essential medicines in a Sub-Saharan country, Ghana.

Rate constants are reported for the pH-independent hydrolysis of 4-methoxyphenyl-2,2-dichloroethanoate in aqueous solution as a function of the concentration of added cyanomethane ( acetonitrile), polyethylene glycol ( PEG 400) and tetrahydrofuran ( THF). The concentration of water was varied between ca. 25 and 55.5 M. It was found that the variation in water activity yields only a minor contribution to the observed variation in rate constants. Interestingly, for both cyanomethane and PEG 400 log(k) varies approximately linearly with the molar concentration of water. Medium effects in highly aqueous solutions ( [ H2O] > 50 M) of ethanol, 1-propanol, 2-propanol, 1-butanol and 2-methyl-2-propanol have also been determined. Unexpectedly, in this concentration range the alcohols induce significantly smaller effects per unit volume than cyanomethane. The present results are discussed in terms of pairwise interaction parameters. Isobaric activation parameters have been determined and reveal remarkable differences in the nature of the induced medium effects.

Overexpression of recombinant proteins in bacteria may lead to their aggregation and deposition in inclusion bodies. Since the conformational properties of proteins in inclusion bodies exhibit many of the characteristics typical of amyloid fibrils. Based on these findings, we hypothesize that the rate at which proteins form amyloid fibrils may be predicted from their propensity to form inclusion bodies. To establish a method based on this concept, we first measured by SDS-PAGE and confocal microscopy the level of inclusion bodies in E. coli cells overexpressing the 40-residue amyloid-beta peptide, Aβ40, wild-type and 24 charge mutants. We then compared these results with a number of existing computational aggregation propensity predictors as well as the rates of aggregation measured in vitro for selected mutants. Our results show a strong correlation between the level of inclusion body formation and aggregation propensity, thus demonstrating the power of this approach and its value in identifying factors modulating aggregation kinetics.

The 2' OH of the peptidyl-tRNA substrate is thought to be important for catalysis of both peptide bond formation and peptide release in the ribosomal active site. The release reaction also specifically depends on a release factor protein (RF) to hydrolyze the ester linkage of the peptidyl-tRNA upon recognition of stop codons in the A site. Here, we demonstrate that certain amino acid substitutions (in particular those containing hydroxyl or thiol groups) in the conserved GGQ glutamine of release factor RF1 can rescue defects in the release reaction associated with peptidyl-tRNA substrates lacking a 2' OH. We explored this rescue effect through biochemical and computational approaches that support a model where the 2' OH of the P-site substrate is critical for orienting the nucleophile in a hydrogen-bonding network productive for catalysis.

Sample preparation represents about two-thirds of the cost of analysis and often presents logistical bottlenecks in analytical and environmental chemistry laboratories, so reducing our capacity and preparedness to quantify organic pollutants rapidly and accurately. Selective pressurized liquid extraction (SPLE) is an analytical technique that builds upon PLE by incorporating matrix-compound (i.e., interference) retainers into the extraction step, so reducing sample-preparation steps and increasing sample throughput. SPLE methods offer distinct advantages over traditional methods, namely reduction in the costs intrinsic to sample preparation (i.e., time, solvents, labor, laboratory space, training, and potential loss of analytes). The ability to analyze and to evaluate rapidly a large number of samples directly increases the analytical capacity and preparedness of a laboratory for certain situations (e.g., large-scale studies or environmental emergencies). We review the analytical improvements via SPLE and its wide-ranging applications.

Over the past 30 years a vast number of studies have demonstrated the presence of pharmaceutical residues in the environment. But still knowledge is scarce regarding the interaction of these emerging pollutants with various matrices in nature. A chromatographic system with on-line detection was developed to perform a sorption study of six selected pharmaceuticals to four natural sediments and dewatered digested sewage treatment plant sludge with differing physicochemical characteristics. Sorption effects, measured as asymmetry factors and recoveries, differed pronouncedly among the pharmaceuticals and between the matrices, which could be explained by basic physicochemical properties of the investigated compounds in relation to matrix characteristics. Protonated and deprotonated molecular properties had the greatest importance for sorbate–sorbent interactions. Atenolol, with cationic properties, showed the highest degree of sorption regardless of the matrix studied. Diclofenac and furosemide, both acids, showed the least tendency towards interactions to natural matrices. Among the neutral compounds bendroflumethiazide, carbamazepine and oxazepam, weaker forces, such as van der Waals, aromatic electron donor–acceptor interactions, and hydrogen forces, seemed more important to determine sorption differences. Results revealed that sorption of pharmaceuticals on natural sediments decreased in the order: atenolol (+) > bendroflumethiazide > oxazepam > carbamazepine > diclofenac (−) > furosemide(–). The matrix content of organic matter measured as total organic carbon (TOC) clearly dictated drug sorption. Beside from studying matrix interaction, these results and the developed technique and methodology might find use in the development of new removal processes of pharmaceuticals from wastewater based on improved knowledge concerning chemical interactions to filter materials.

In environmental trace analysis there is often a need to enrich the compounds of interest from a large sample volume, where the use of solid phase extraction (SPE) is more or less the standard technique. The presence of the sample matrix can cause clogging of the SPE-column, especially at the end of a sample load. Swedish surface waters are often humic rich making the use of traditional sample loading by a vacuum manifold very limited. This obstacle forced the development of a different sample loading technique, based on compressed air and sand as an in-line-filter, designed to load larger sample volumes as needed in trace level analysis of hormones in surface water. The developed technique, combined with a UPLC MS/MS method, showed promising reproducibility and accuracy, and enabled increased sensitivity for the analysis of hormones in humic rich surface water.

Thermal degradation of antibiotics has been studied for decades in a broad range of disciplines including food production, agriculture and analytical chemistry. Yet, there is a lack of thermal stability data for many antibiotics. Here we systematically investigated the thermal stability of ten commonly prescribed antibiotics applying a laborsaving automated inhouse pressurized dynamic flow-through system. The design of the system allowed a fast access to a large number of data at medium to subcritical water temperatures, ranging from 50-250 °C. The five ß-lactams cefadroxil, cefuroxime, amoxicillin, penicillin V, and penicillin G showed a high degree of stability with a maximum degradation of less than 30 % at 150 °C. The two quinolones ciprofloxacin and norfloxacin showed a very high thermal stability up to 200 °C, as did trimethoprim andsulfamethoxazole. At 250 °C all antibiotics were either partly of fully removed. The tetracycline doxycycline showed a specific removal pattern probably involving both binding to metal surfaces at lower temperatures as well as degradation at increased temperatures.