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  • 1.
    Assarsson, A.
    et al.
    Lund University.
    Nasir, I.
    Lund University.
    Lundqvist, M.
    Lund University.
    Cabaleiro-Lago, Celia
    Lund Univeristy.
    Kinetic and thermodynamic study of the interactions between human carbonic anhydrase variants and polystyrene nanoparticles of different size2016Inngår i: RSC Advances, ISSN 2046-2069, E-ISSN 2046-2069, Vol. 6, nr 42, s. 35868-35874Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    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.

  • 2.
    Assarsson, Anna
    et al.
    Lund University.
    Hellstrand, Erik
    Lund University.
    Cabaleiro-Lago, Celia
    Lund University.
    Linse, Sara
    Lund University.
    Charge dependent retardation of amyloid β aggregation by hydrophilic proteins2014Inngår i: ACS Chemical Neuroscience, ISSN 1948-7193, E-ISSN 1948-7193, Vol. 5, nr 4, s. 266-74Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    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.

  • 3.
    Assarsson, Anna
    et al.
    Lund University.
    Linse, Sara
    Lund University.
    Cabaleiro-Lago, Celia
    Lund University.
    Effects of polyamino acids and polyelectrolytes on amyloid β fibril formation2014Inngår i: Langmuir, ISSN 0743-7463, E-ISSN 1520-5827, Vol. 30, nr 29, s. 8812-8Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    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.

  • 4.
    Assarsson, Anna
    et al.
    Lund University.
    Pastoriza-Santos, Isabel
    Spain.
    Cabaleiro-Lago, Celia
    Lund University.
    Inactivation and adsorption of human carbonic anhydrase II by nanoparticles2014Inngår i: Langmuir, ISSN 0743-7463, E-ISSN 1520-5827, Vol. 30, nr 31, s. 9448-56Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    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.

  • 5.
    Bechshoft, Thea
    et al.
    Danmark.
    Wright, Andrew J.
    Danmark.
    Weisser, Johan J.
    Danmark.
    Teilmann, Jonas
    Danmark.
    Dietz, Rune
    Danmark.
    Hansen, Martin
    Danmark.
    Björklund, Erland
    Danmark.
    Styrishave, Bjarne
    Danmark.
    Developing a new research tool for use in free-ranging cetaceans: recovering cortisol from harbour porpoise skin2015Inngår i: Conservation Physiology, E-ISSN 2051-1434, Vol. 3Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    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.

  • 6.
    Cabaleiro-Lago, Celia
    et al.
    Irland.
    Quinlan-Pluck, Fiona
    Irland.
    Lynch, Iseult
    Irland.
    Dawson, Kenneth A
    Irland.
    Linse, Sara
    Lund University.
    Dual effect of amino modified polystyrene nanoparticles on amyloid β protein fibrillation2010Inngår i: ACS Chemical Neuroscience, ISSN 1948-7193, E-ISSN 1948-7193, Vol. 1, nr 4, s. 279-87Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    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.

  • 7.
    Cabaleiro-Lago, Celia
    et al.
    Irland.
    Quinlan-Pluck, Fiona
    Irland.
    Lynch, Iseult
    Irland.
    Lindman, Stina
    Lund University.
    Minogue, Aedin M
    Irland.
    Thulin, Eva
    Lund University.
    Walsh, Dominic M
    Irland.
    Dawson, Kenneth A
    Irland.
    Linse, Sara
    Lund University.
    Inhibition of amyloid beta protein fibrillation by polymeric nanoparticles2008Inngår i: Journal of the American Chemical Society, ISSN 0002-7863, E-ISSN 1520-5126, Vol. 130, nr 46, s. 15437-43Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    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.

  • 8.
    Eberson, Lennart
    et al.
    Department of Chemistry, Chemical Physics, Lund.
    Persson, Ola
    Department of Chemistry, Chemical Physics, Lund.
    Hall Jr, H. K.
    Padias, A. B.
    Steel, P. J.
    Spin trapping of radicals from the reactions between donor and acceptor olefins:  further evidence for the tetramethylene diradical intermediate as the initiator of spontaneous copolymerization2000Inngår i: Macromolecules, ISSN 0024-9297, E-ISSN 1520-5835, Vol. 33, nr 6, s. 2021-2029Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    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.

  • 9.
    Hakansson, Andreas
    et al.
    Högskolan Kristianstad, Sektionen för lärande och miljö, Avdelningen för Humanvetenskap.
    Askaner, Måns
    Kristianstad University.
    Innings, Fredrik
    Tetra Pak, Lund.
    Extent and mechanism of coalescence in rotor-stator mixer food-emulsion emulsification2016Inngår i: Journal of Food Engineering, ISSN 0260-8774, E-ISSN 1873-5770, Vol. 175, s. 127-135Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    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.

  • 10.
    Hallgren, Pär
    et al.
    Högskolan Kristianstad, Sektionen för lärande och miljö.
    Mårtensson, Lennart
    Högskolan Kristianstad, Sektionen för lärande och miljö.
    Mathiasson, Lennart
    Division of Analytical Chemistry, Lund University.
    A new spectrophotometric method for improved indirect measurement of low levels of vitellogenin using malachite green2012Inngår i: International Journal of Environmental Analytical Chemistry, ISSN 0306-7319, E-ISSN 1029-0397, Vol. 92, nr 7, s. 894-908Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    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.

  • 11.
    Hallgren, Pär
    et al.
    Högskolan Kristianstad, Sektionen för Lärarutbildning.
    Mårtensson, Lennart
    Högskolan Kristianstad, Sektionen för Lärarutbildning.
    Mathiasson, Lennart
    Division of Analytical Chemistry, Lund University.
    Improved spectrophotometric vitellogenin determination via alkali-labile phosphate in fish plasma: a cost effective approach for assessment of endocrine2009Inngår i: International Journal of Environmental Analytical Chemistry, ISSN 0306-7319, E-ISSN 1029-0397, Vol. 89, nr 14, s. 1023-1042Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    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.

  • 12.
    Hansen, M.
    et al.
    University of California Berkeley.
    Popovic, O.
    University of Copenhagen.
    Björklund, Erland
    Högskolan Kristianstad, Sektionen för lärande och miljö, Avdelningen för Naturvetenskap.
    Krogh, K.
    University of Copenhagen.
    Stoumann, L.
    University of Copenhagen.
    Jacobsen, C.S.
    Geological Survey of Denmark and Greenland.
    Halling-Sørensen, B.
    University of Copenhagen.
    Impact of animal manure separation technologies on steriod hormone distribution: consequences for agricultural practices2014Konferansepaper (Fagfellevurdert)
    Abstract [en]

    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.

  • 13.
    Hansen, Martin
    et al.
    Faculty of Health and Medical Sciences, University of Copenhagen.
    Björklund, Erland
    Faculty of Health and Medical Sciences, University of Copenhagen.
    Popovic, Olga
    Faculty of Science, University of Copenhagen.
    Jensen, Lars S.
    Faculty of Science, University of Copenhagen.
    Jacobsen, Carsten S.
    Geological Survey of Denmark and Greenland (GEUS).
    Sedlak, David L.
    Department of Civil and Environmental Engineering, University of California, Berkeley.
    Halling-Sørensen, Bent
    Faculty of Health and Medical Sciences, University of Copenhagen.
    Animal manure separation technologies diminish the environmental burden of steroid hormones2015Inngår i: Environmental Science and Technology Letters, E-ISSN 2328-8930, Vol. 2, nr 4, s. 133-137Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    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.

  • 14.
    Hansen, Martin
    et al.
    Institut for Farmaci, Københavns Universitet.
    Rodríguez-Navas, Carlos
    University of Texas Southwestern Medical Center.
    Björklund, Erland
    Högskolan Kristianstad, Sektionen för lärande och miljö, Avdelningen för Naturvetenskap.
    Lægemidler i vandmiljøet på Mallorca2013Inngår i: Dansk kemi, ISSN 0011-6335, Vol. 94, nr 8, s. 24-27Artikkel i tidsskrift (Annet (populærvitenskap, debatt, mm))
  • 15.
    Heydorn, Per
    Högskolan Kristianstad, Fakulteten för naturvetenskap.
    Inbreeding decreases upwind pheromone: mediated male flight and frequency in female calling behavior in a lab culture of the pyraloid moth Plodia interpunctella2018Independent thesis Basic level (degree of Bachelor), 10 poäng / 15 hpOppgave
    Abstract [en]

    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.

  • 16.
    Håkansson, Andreas
    Högskolan Kristianstad, Sektionen för lärande och miljö, Avdelningen för Praktisk-estetiska ämnen.
    Droplet breakup in high-pressure homogenizers2015Inngår i: Engineering aspects of food emulsification and homogenization / [ed] Marilyn Rayner and Petr Dejmek, Boca Raton: Taylor & Francis Group, 2015, s. 125-148Kapittel i bok, del av antologi (Annet vitenskapelig)
    Abstract [en]

    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.

  • 17.
    Johansson, Magnus
    et al.
    Uppsala University.
    Ieong, Ka-Weng
    Uppsala University.
    Trobro, Stefan
    Uppsala University.
    Strazewski, Peter
    Frankrike.
    Åqvist, Johan
    Uppsala University.
    Pavlov, Michael Y.
    Uppsala University.
    Ehrenberg, Måns
    Uppsala University.
    pH-sensitivity of the ribosomal peptidyl transfer reaction dependent on the identity of the A-site aminoacyl-tRNA2011Inngår i: Proceedings of the National Academy of Sciences of the United States of America, ISSN 0027-8424, E-ISSN 1091-6490, Vol. 108, nr 1, s. 79-84Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    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.

  • 18.
    Lundqvist, Martin
    et al.
    Lund University.
    Cabaleiro-Lago, Celia
    Lund University.
    Buffer formulation affects the interaction between lysozyme and polymeric nanoparticles2017Inngår i: Journal of Colloid and Interface Science, ISSN 0021-9797, E-ISSN 1095-7103, Vol. 504, s. 78-85Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    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.

  • 19.
    Mathiasson, Lennart
    et al.
    Lund University.
    Mårtensson, Lennart
    Högskolan Kristianstad, Sektionen för lärande och miljö, Avdelningen för Naturvetenskap. Högskolan Kristianstad, Forskningsmiljön Man & Biosphere Health (MABH).
    Adequate sampling, an important step towards reiable decisions based on analytical results2010Inngår i: Proceedings: Linnaeus ECO-TECH´10, Kalmar,  Sweden, Nov 22-24, 2010, 2010, s. 16-23Konferansepaper (Fagfellevurdert)
    Abstract [en]

    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

  • 20.
    Nasir, Irem
    et al.
    Lund University.
    Lundqvist, Martin
    Lund University.
    Cabaleiro-Lago, Celia
    Lund University.
    Size and surface chemistry of nanoparticles lead to a variant behavior in the unfolding dynamics of human carbonic anhydrase2015Inngår i: Nanoscale, ISSN 2040-3364, E-ISSN 2040-3372, Vol. 7, nr 41, s. 17504-15Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    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.

  • 21.
    Nielsen, Frederik Knud
    et al.
    Danmark.
    Hansen, Cecilie Hurup
    Danmark.
    Fey, Jennifer Anna
    Danmark.
    Hansen, Martin
    USA.
    Halling-Sørensen, Bent
    Danmark.
    Björklund, Erland
    Högskolan Kristianstad, Sektionen för lärande och miljö, Avdelningen för Naturvetenskap.
    Styrishave, Bjarne
    Danmark.
    Mixture effects of 3 mechanistically different steroidogenic disruptors (prochloraz, genistein, and ketoconazole) in the H295R cell assay2015Inngår i: International journal of toxicology, ISSN 1091-5818, E-ISSN 1092-874X, Vol. 34, nr 6, s. 534-542Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    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.

  • 22.
    Oppong Bekoe, Samuel
    et al.
    Faculty of Health and Medical Sciences, University of Copenhagen.
    Bak, Søren Alex
    Faculty of Health and Medical Sciences, University of Copenhagen.
    Björklund, Erland
    Faculty of Health and Medical Sciences, University of Copenhagen.
    Krogh, Kristine A.
    Faculty of Health and Medical Sciences, University of Copenhagen.
    Okine, Nathaniel N. N. A.
    Kwame Nkrumah University of Science and Technology, Kumasi, Ghana.
    Adosraku, Reimmiel K.
    Kwame Nkrumah University of Science and Technology, Kumasi, Ghana.
    Styrishave, Bjarne
    Faculty of Health and Medical Sciences, University of Copenhagen.
    Hansen, Martin
    Faculty of Health and Medical Sciences, University of Copenhagen.
    Determination of thirteen antibiotics in drug products: a new LC-MS/MS tool for screening drug product quality2014Inngår i: Analytical Methods, ISSN 1759-9660, E-ISSN 1759-9679, Vol. 6, s. 5847-5855Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    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.

  • 23.
    Shaw, Jeffrey J.
    et al.
    Johns Hopkins University School of Medicine, Baltimore.
    Trobro, Stefan
    Institutionen för Cell och Molekylärbiologi, Uppsala Universitet.
    He, Shan L.
    Johns Hopkins University School of Medicine, Baltimore.
    Åqvist, Johan
    Institutionen för Cell och Molekylärbiologi, Uppsala Universitet.
    Green, Rachel
    Johns Hopkins University School of Medicine, Baltimore.
    A Role for the 2' OH of peptidyl-tRNA substrate in peptide release on the ribosome revealed through RF-mediated rescue2012Inngår i: Chemistry and Biology, ISSN 1074-5521, E-ISSN 1879-1301, Vol. 19, nr 8, s. 983-993Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    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.

  • 24.
    Subedi, Bikram
    et al.
    Baylor University, Waco, USA.
    Aguilar, Lissette
    Baylor University, Waco, USA.
    Robinson, Eleanor M.
    Baylor University, Waco, USA.
    Hageman, Kimberley J.
    University of Otago, New Zealand.
    Björklund, Erland
    Högskolan Kristianstad, Sektionen för lärande och miljö, Avdelningen för Naturvetenskap.
    Sheesley, Rebecca J.
    Baylor University, Waco, USA.
    Usenko, Sascha
    Baylor University, Waco, USA.
    Selective pressurized liquid extraction as a sample-preparation technique for persistent organic pollutants and contaminants of emerging concern2015Inngår i: TrAC. Trends in analytical chemistry, ISSN 0165-9936, E-ISSN 1879-3142, Vol. 68, s. 119-132Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    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.

  • 25.
    Svahn, Ola
    et al.
    Högskolan Kristianstad, Sektionen för lärande och miljö, Avdelningen för Naturvetenskap. Högskolan Kristianstad, Plattformen för molekylär analys.
    Björklund, Erland
    Högskolan Kristianstad, Sektionen för lärande och miljö, Avdelningen för Naturvetenskap. Högskolan Kristianstad, Plattformen för molekylär analys.
    Describing sorption of pharmaceuticals to lake and river sediments, and sewage sludge from UNESCO Biosphere Reserve Kristianstads Vattenrike by chromatographic asymmetry factors and recovery measurements2015Inngår i: Journal of Chromatography A, ISSN 0021-9673, E-ISSN 1873-3778, Vol. 415, s. 73-82Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    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.

  • 26.
    Svahn, Ola
    et al.
    Högskolan Kristianstad, Sektionen för lärande och miljö, Avdelningen för Naturvetenskap. Högskolan Kristianstad, Plattformen för molekylär analys.
    Björklund, Erland
    Högskolan Kristianstad, Sektionen för lärande och miljö, Avdelningen för Naturvetenskap. Högskolan Kristianstad, Plattformen för molekylär analys.
    Thermal stability assessment of antibiotics in moderate temperature and subcriticalwater using a pressurized dynamic flow-through system2015Inngår i: International Journal of Innovation and Applied Studies, ISSN 2028-9324, Vol. 11, nr 4, s. 872-880Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    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.

  • 27.
    Svensson, Britt-Marie
    Högskolan Kristianstad, Sektionen för Lärarutbildning.
    Methodology for evaluation of hazards from solid waste and landfill-generated leachate2008Doktoravhandling, monografi (Annet vitenskapelig)
    Abstract [en]

    A methodology based on an analytical protocol for evaluation of hazards from landfill leachate and solid waste is described. A dynamic analytical protocol, the LAQUA protocol, including measurements of inorganic and water-quality parameters, polar and non-polar organic marker compounds, and toxicity, was constructed. An acute toxicity test, using the brackish water crustacean Artemia salina as test organism, was developed. The methodology was applied to authentic problems such as investigation of different treatment techniques for landfill leachate, evaluation of leaching tests and characterization of solid wastes, and an investigation of a filter material aimed for leachate treatment. Investigated treatment methods comprised in all cases pre-treatment by aeration combined with sedimentation, followed by one of: bioremediation, ozonation, chemical oxidation by Fenton’s reagent, or geo-bed filters. Evaluated filter materials were mixtures of natural or residual waste products. A combination of pre-treatment followed by a geo-bed filter containing a mixture of peat and carbon-containing ash gave an efficient simultaneous removal of metals and organic pollutants. The performance of two leaching tests for characterization of solid waste, the up-flow percolation test (SIS-CEN/TS 14405:2004) and the batch test (SS-EN 12457 -3), was investigated. Solid waste materials (sludge from street gutters and fragmented metallic waste) were characterized using leaching tests and the hazards of the materials were evaluated in the eluate, obtained at specific liquid-to-solid ratios (L/S). The L/S 2 and L/S 10 values were compared with limit values included in the waste acceptance criteria (WAC). The analyses were extended towards specific organic compounds, such as individual phenolic compounds and polychlorinated biphenyls (PCB). Organic compounds were found in eluates from both types of tests, showing the possibility to use these methods to evaluate the leaching of such compounds from waste materials. The use of authentic leachate as leachant, leads to increased concentrations of heavy metals in the eluate, compared to the prescribed use of demineralised water as leachant. Generally good agreement was found between the results of the two leaching methods. A strategy based on batch tests is described for investigation of a filter material for leachate treatment. Batch tests gave suitable information about the leaching from new and used material, and showed high removal efficiencies of metals and non-polar organic compounds. However, for investigation of removal of polar organic markers (e.g. phenolic compounds) a batch test is not sufficient and should be supplemented by a column test.

  • 28.
    Tavahodi, Mojtaba
    et al.
    Iran.
    Schulz, Christopher
    Lund University.
    Assarsson, Anna
    Lund University.
    Ortiz, Roberto
    Lund University.
    Ludwig, Roland
    Austria.
    Cabaleiro-Lago, Celia
    Lund University.
    Haghighi, Behzad
    Iran.
    Gorton, Lo
    Lund University.
    Interaction of polymer-coated gold nanoparticles with cellobiose dehydrogenase: the role of surface charges2018Inngår i: Journal of Electroanalytical Chemistry, ISSN 1572-6657, Vol. 819, s. 226-233Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    Studying the interaction of functional proteins such as enzymes and nanoparticles (NPs) includes the important topic of investigating any possible changes in stability and function of enzymes in nanostructured environments. The effects of NPs on the enzyme activity and stability are governed by their physical and chemical properties such as structure, shape, size, surface chemistry and their surface charges. In this study, the influence of negatively and positively charged AuNPs are investigated on the activity of immobilized Myriococcum thermophilum cellobiose dehydrogenase (MtCDH) and its electron transfer rate with graphite electrodes modified with positively and negatively AuNPs. The MtCDH modified graphite electrode premodified with positively charged AuNPs showed an alkaline shift in the pH of maximum activity from pH5.5 to 8. No change in the pH of maximum activity was observed when MtCDH graphite electrodes were premodified with negatively charged AuNPs. The results clearly demonstrated the effect of surface charge of AuNPs on the activity of the enzyme. The catalytic current density and the KMapp value for MtCDH graphite electrode premodified with positively charged AuNPs were enhanced with up to 66 and 8 times, respectively. Two spectroscopic assays were also performed in solution to investigate the influence of the presence of positively or negatively charged AuNPs on the activity of MtCDH in homogeneous solution. The results clearly demonstrated that not only the rate of the heterogeneous electron transfer between the immobilized MtCDH and the electrode but also the rate of the homogeneous electron transfer between soluble MtCDH and the acceptor was highly dependent on the type of surface charge of the AuNPs.

  • 29.
    Trobro, Stefan
    et al.
    Uppsala University.
    Åqvist, Johan
    Uppsala University.
    Mechanism of the translation termination reaction on the ribosome2009Inngår i: Biochemistry, ISSN 0006-2960, E-ISSN 1520-4995, Vol. 48, nr 47, s. 11296-303Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    Ribosomal release factors (RFs) catalyze the termination of protein synthesis by triggering hydrolysis of the peptidyl-tRNA ester bond in the peptidyl transferase center of the ribosome. With new medium-resolution crystallographic structures of RF-ribosome complexes available, it has become possible to examine the detailed mechanism of this process to resolve the key factors responsible for catalysis of the termination reaction. Here, we report computer simulations of the termination reaction that utilize both the new RF complex structures and information from a high-resolution complex with a P-site substrate analogue. The calculations yield a consistent reaction mechanism that reproduces experimental rates and allows us to identify key interactions responsible for the catalytic efficiency. The results are also in general agreement with an earlier model based on molecular docking. The methylated glutamine residue of the universally conserved GGQ motif plays a key role in the hydrolysis reaction by orienting the water nucleophile and by stabilizing the transition state, and its side chain makes an entropic contribution to the lowering of the activation barrier. Two additional water molecules interacting with the P-site substrate are also found to be critically important. Furthermore, the 2'-OH group of the peptidyl-tRNA substrate is predicted to act as a proton shuttle for the leaving group in analogy with the consensus mechanism for peptidyl transfer. Thus, the ribosome's ability to catalyze both the termination (hydrolysis) and peptidyl transfer (aminolysis) reactions is largely explained by this type of unified mechanism, with similar transition states occurring in both processes.

  • 30.
    Wu, Shiying
    et al.
    Department of Cell and Molecular Biology, Uppsala University.
    Chen, Yu
    Department of Cell and Molecular Biology, Uppsala University.
    Mao, Guanzhong
    Department of Cell and Molecular Biology, Uppsala University.
    Trobro, Stefan
    Department of Cell and Molecular Biology, Uppsala University.
    Kwiatkowski, Marek
    Department of Cell and Molecular Biology, Uppsala University.
    Kirsebom, Leif A.
    Department of Cell and Molecular Biology, Uppsala University.
    Transition-state stabilization in Escherichia coli ribonuclease P RNA-mediated cleavage of model substrates2014Inngår i: Nucleic Acids Research, ISSN 0305-1048, E-ISSN 1362-4962, Vol. 42, nr 1, s. 631-642Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    We have used model substrates carrying modified nucleotides at the site immediately 5' of the canonical RNase P cleavage site, the -1 position, to study Escherichia coli RNase P RNA-mediated cleavage. We show that the nucleobase at -1 is not essential but its presence and identity contribute to efficiency, fidelity of cleavage and stabilization of the transition state. When U or C is present at -1, the carbonyl oxygen at C2 on the nucleobase contributes to transition-state stabilization, and thus acts as a positive determinant. For substrates with purines at -1, an exocyclic amine at C2 on the nucleobase promotes cleavage at an alternative site and it has a negative impact on cleavage at the canonical site. We also provide new insights into the interaction between E. coli RNase P RNA and the -1 residue in the substrate. Our findings will be discussed using a model where bacterial RNase P cleavage proceeds through a conformational-assisted mechanism that positions the metal(II)-activated H2O for an in-line attack on the phosphorous atom that leads to breakage of the phosphodiester bond.

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