Ethylene oxide and propylene oxide random copolymer/sodium chloride aqueous two-phase systems: wetting and adsorption on dodecyl-agarose and polystyrene

Liquid/liquid partition chromatography is a mild yet powerful separation method for a variety of biological materials. This work demonstrates that it should be feasible to immobilize an ethylene oxide-propylene oxide (EO/PO) random copolymer solution and to use a solution of NaCl equilibrated against the polymer solution as the mobile-phase (poly (EO-PO) [P(EO-PO)] and NaCl form two aqueous phases known as aqueous two-phase systems). Three random copolymers with different molecular weights and EO/PO ratios were used. Dodecyl-agarose and polystyrene were tested as possible supports. The wetting energies of the aqueous two-phase systems on these two kinds of surfaces were calculated as well as contact angles for each phase on the same surfaces. Finally, the thickness of P(EO-PO) adsorption layers on polystyrene lattices were measured by dynamic light scattering. Contact angle measurements indicate that indeed some EO/PO copolymers preferentially wet hydrophobic substrates, forming thin films.     

Genotoxic effects of ethylene oxide and propylene oxide: a comparative study

The two alkylating agents ethylene oxide (EO) and propylene oxide (PO) were compared for genotoxic effectiveness in various test systems. The study was undertaken partly to shed light on the difference between the compounds found after chronic exposure of monkeys (Lynch et al., 1984) where EO but not PO was able to induce SCE and chromosomal aberrations. In the present study EO was found to be 5–10 times more effective than PO with respect to gene conversion and reverse mutation in Saccharomyces cerevisiae D7 and sister-chromatid conversion in S. cerevisiae RS112. In contrast, the abilities of the two compounds to induce point mutation in S. typhimurium strains and SCE in human lymphocytes were approximately equal. One possible cause of EO being more effective than PO in certain respects, discussed on the basis of inference from earlier studies, is an expected difference in ability to cause strand breaks via alkylation of DNA-phosphate groups.     

Effect of salts and surfactants on the partitioning of Fusarium solani pisi cutinase in aqueous two-phase systems of thermoseparating ethylene oxide/propylene oxide random copolymer and hydroxypropyl starch

An aqueous two-phase system composed by a thermoseparating random copolymer of ethylene oxide/propylene oxide 50/50 (%w/w), Breox, and hydroxypropyl starch – Reppal PES 100 was evaluated for the partitioning of Fusarium solani pisi recombinant cutinase. The effect of several additives on the partitioning of pure cutinase was evaluated. Micelles of sodium dodecanoate provided a ten-fold increase of the partitioning coefficient (K=9) and recovery yields of 60-75%. The phase diagrams of the systems composed of Breox, Reppal and sodium dodecanoate were determined and it was found that in systems with high surfactant concentrations, the binodal was moved to lower polymer concentrations, enabling a two-phase system with 6% (w/w) of each polymer.     

Why iron–dithiocarbamates ensure detection of nitric oxide in cells and tissues

The in vivo mechanism of NO trapping by iron-dithiocarbamate complexes is considered. Contrary to common belief, we find that in biological systems the NO radicals are predominantly trapped by ferric iron-dithiocarbamates. Therefore, the trapping leads to ferric mononitrosyl complexes which are diamagnetic and cannot be directly detected with Electron Paramagnetic Resonance spectroscopy. The ferric mononitrosyl complexes are far easily reduced to ferrous state with L-cysteine, glutathione, ascorbate or dithiocarbamate ligands than their non-nitrosyl counterpart. When trapping NO in oxygenated biological systems, the majority of trapped nitric oxide is found in diamagnetic ferric mononitrosyl iron complexes. Only a minority fraction of NO is trapped in the form of paramagnetic ferrous mononitrosyl iron complexes with dithiocarbamate ligands. Subsequent ex vivo reduction of biological samples sharply increases the total yield of the paramagnetic mononitrosyl iron complexes. Reduction also eliminates the overlapping EPR spectrum from Cu(2+)-dithiocarbamate complexes. This facilitates the quantification of yields from NO trapping.     

Cutinase purification on poly(ethylene glycol)–hydroxypropyl starch aqueous two-phase systems

The partition behaviour of cutinase on poly(ethylene glycol) (PEG)–hydroxypropyl starch aqueous two-phase systems was characterized. The effect of molecular mass of PEG, the pH of the system and tie-line length on cutinase partition coefficient and cutinase yield to the top phase was investigated for systems prepared with a purified hydroxypropyl starch (Reppal PES 100) and a crude one (HPS). The effect of the presence of different salts, such as sodium chloride, sodium sulphate and ammonium sulphate, on cutinase partition was also studied. The results lead to the conclusion that aqueous two-phase systems composed of PEG and hydroxypropyl starch are not efficient in the purification of cutinase. In the majority of cases, the partition coefficients were very close to 1, with pH being the factor which affects most cutinase partition. Partition coefficients were significantly improved when salts were added to the systems. For PEG 4000–Reppal PES 100 [at pH 4.0; 0.5 M (NH₄)₂SO₄], the partition coefficient for cutinase was 3.7, while a value of 12 was obtained for PEG 4000–HPS (at pH 4.0; 1 M NaCl). An isoelectric point (pI) of 7.8 was confirmed for cutinase by constructing a cross partition graphic from the results obtained in the experiments with different salts.     

Effect of potential amine prodrugs of selective neuronal nitric oxide synthase inhibitors on blood–brain barrier penetration

Several prodrug approaches were taken to mask amino groups in two potent and selective neuronal nitric oxide synthase (nNOS) inhibitors containing either a primary or secondary amino group to lower the charge and improve blood–brain barrier (BBB) penetration. The primary amine was masked as an azide and the secondary amine as an amide or carbamate. The azide was not reduced to the amine under a variety of in vitro and ex vivo conditions. Despite the decrease in charge of the amino group as an amide and as carbamates, BBB penetration did not increase. It appears that the uses of azides as prodrugs for primary amines or amides and carbamates as prodrugs for secondary amines are not universally effective for CNS applications.     

Impact of surfactants on solubilization and activity of the carotenoid cleavage dioxygenase,AtCCD1, in an aqueous micellar reaction system

The effect of various surfactants on both the solubilization of the carotenoid cleavage dioxygenase, AtCCD1, from cell lysates and the enzymatic activity in an aqueous micellar system was investigated. Solubilization with sodium cholate more than doubled the specific activity. Lag phases were observed when Tween surfactants were used for substrate delivery and were dependent on the surfactant and enzyme modification. In contrast to His₆- and GST-tagged AtCCD1, unmodified AtCCD1 showed a 45% increased maximum rate in the Tween 20 system compared to Triton X-100 based reference system. The results emphasize the importance of engineering the interface for the in vitro application of this enzyme family.     

Synthesis and characterization of a novel MPEG–chitosan diblock copolymer and self-assembly of nanoparticles

In this paper, a simple and novel method based on free-radical polymerization initiated by potassium persulfate (KPS) was developed to synthesize the MPEG–chitosan diblock copolymer (MPEG–CS). The obtained MPEG–CS diblock copolymer was characterized by Fourier transform infrared (FTIR), ¹H nuclear magnetic resonance (¹H NMR), X-ray diffraction (XRD) and differential scanning calorimetry (DSC), respectively. The MPEG–CS copolymer could self-assemble into nanoparticles in aqueous solution. A typical TEM photography indicated that the well-spherical nanoparticles with diameter at about 200 nm were obtained. In vitro cell culture assay indicated that MPEG–CS nanoparticles are non-toxic and cell-compatible as the polymer concentration was smaller than 0.6 mg/ml. In conclusion, the obtained MPEG–CS nanoparticles might have great potential application in drug-delivery system.     

Effect of surfactants on the thermal, conformational and rheological properties of collagen

Collagen is an important biomaterial and its interaction with surfactant is important in light of its use in various cosmetics and dermatological applications. Presently, the effect of surfactants on the physico-chemical properties of collagen has been studied. The thermal stability of collagen is reduced by sodium dodecyl sulfate and hexadecyltrimethylammmonium bromide, whereas Triton X-100 does not. The viscosity of collagen is influenced greatly depending on the surfactant concentrations. The secondary structure of collagen shows changes only in the molar ellipticity. The role of charge and concentration of surfactants in influencing the various physico-chemical properties of collagen has been elucidated.     

Cathodic electrochemiluminescence of Ru(bpy)/Nafion coated on graphite oxide electrode in purely aqueous solution

Two cathodic electrochemiluminescence (ECL) peaks have been obtained at ?0.99 and ?1.80 V (vs. SCE), respectively, from Ru(bpy)/Na?on coated onto a graphite oxide electrode in purely aqueous solution under cyclic voltammetric (CV) conditions, without addition of any reducing or oxidative reagents. These two ECL peaks were found to correlate to initial scan direction, pH, and reversal potential. Na?on played an important role in the generation of these two ECL peaks because no cathodic emission was observed in the system without Na?on. It seems that a part of Ru(bpy) electrogenerated at positive potential can remain in the Na?on, even at negative potentials. It was con?rmed that Ru(bpy)⁺ was formed at ?1.80 V by addition of S₂O. The ECL peak at ?0.99 V is attributed to the reaction of Ru(bpy)³⁺ and OH^?. The ECL peak at ?1.80 V is probably due to the annihilation reaction of Ru(bpy)³⁺ and Ru(bpy)⁺. Copyright © 2003 John Wiley & Sons, Ltd.     

Optimization of androstenedione production in an organic–aqueous two-liquid phase system

A systematic evaluation of the effect of key operational parameters on the selective cleavage of sitosterol to 4-androstene-3,17-dione (AD) with Mycobacterium sp. NRRL B-3805 in a dioctyl phthalate: aqueous buffer two-liquid phase system was performed. Of the parameters assessed, buffer composition, biomedium pH, temperature, and biomass and substrate concentration were those that mostly affected overall bioconversion rate. The optimum pH was 7.5 with Tris buffer. The highest bioconversion rate was observed at 35 °C, although at 40 °C bioconversion activity was virtually lost. Michaelis–Menten type kinetics adequately described the bioconversion system. Increasing biomass concentration from 10 to 70 g_{wet cell weight} l⁻¹ favored AD final yield, although the specific AD yield slightly decreased.     

Interaction between Shaoyao–Gancao–Tang and a laxative with respect to alteration of paeoniflorin metabolism by intestinal bacteria in rats

Shaoyao-Gancao-Tang (SGT), a traditional Chinese herbal medicine (Kampo formulation) containing Shaoyao (Paeoniae Radix) and Gancao (Glycyrrhizae Radix), is co-administered with laxative sodium picosulfate as a premedication for relieving the pain accompanying colonoscopy. Paeoniflorin (PF), an active glycoside of SGT, is metabolized into the antispasmodic agent paeonimetabolin-I (PM-I) by intestinal bacteria after oral administration. The objective of the present study was to investigate whether the co-administered laxative (sodium picosulfate) influences the metabolism of PF to PM-I by intestinal bacteria. We found that the PF-metabolizing activity of intestinal bacteria in rat feces was significantly reduced to approximately 34% of initial levels by a single sodium picosulfate pretreatment and took approximately 6 days to recover. Repeated administration of SGT after the sodium picosulfate pretreatment significantly shortened the recovery period to around 2 days. Similar results were also observed for plasma PM-I concentration. Since PM-I has muscle relaxant activity, the present results suggest that repetitive administration of SGT after sodium picosulfate pretreatment might be useful to relieve the pain associated with colonoscopy.     

Role of nitric oxide in obsessive–compulsive behavior and its involvement in the anti-compulsive effect of paroxetine in mice

In view of the reports that nitric oxide modulates the neurotransmitters implicated in obsessive–compulsive disorder, patients with obsessive–compulsive disorder exhibit higher plasma nitrate levels, and drugs useful in obsessive–compulsive disorder influence nitric oxide, we hypothesized that nitric oxide may have some role in obsessive–compulsive behavior. We used marble-burying behavior of mice as the animal model of obsessive–compulsive disorder, and nitric oxide levels in brain homogenate were measured using amperometric nitric oxide-selective sensor method. Intraperitoneal administration of nitric oxide enhancers viz. nitric oxide precursor—l-arginine (800 mg/kg), nitric oxide donor—sodium nitroprusside (3 mg/kg) or phosphodiesterase type 5 inhibitor—sildenafil (3 mg/kg) significantly increased marble-burying behavior as well as brain nitrites levels, whereas treatment with 7-nitroindazole—neuronal nitric oxide synthase inhibitor (20–40 mg/kg, i.p.) or paroxetine—selective serotonin reuptake inhibitor (5–10 mg/kg, i.p.) dose dependently attenuated marble-burying behavior and nitrites levels in brain. Further, co-administration of sub-effective doses of 7-nitroindazole (10 mg/kg) and paroxetine (2.5 mg/kg) significantly attenuated marble-burying behavior. Moreover, pretreatment with l-arginine (400 mg/kg, i.p.), sodium nitroprusside (2.0 mg/kg, i.p.) or sildenafil (2.0 mg/kg, i.p.) significantly attenuated the inhibitory influence of 7-nitroindazole (40 mg/kg) or paroxetine (10 mg/kg) on marble-burying behavior as well as on brain nitrites levels. None of the above treatment had any significant influence on locomotor activity. In conclusion, obsessive compulsive behavior in mice appears related to nitric oxide in brain, and anti-compulsive effect of paroxetine appears to be related to decrease central levels of nitric oxide.     

Combining capture–recapture data and pedigree information to assess heritability of demographic parameters in the wild

Quantitative genetic analyses have been increasingly used to estimate the genetic basis of life‐history traits in natural populations. Imperfect detection of individuals is inherent to studies that monitor populations in the wild, yet it is seldom accounted for by quantitative genetic studies, perhaps leading to flawed inference. To facilitate the inclusion of imperfect detection of individuals in such studies, we develop a method to estimate additive genetic variance and assess heritability for binary traits such as survival, using capture–recapture (CR) data. Our approach combines mixed‐effects CR models with a threshold model to incorporate discrete data in a standard ‘animal model’ approach. We employ Markov chain Monte Carlo sampling in a Bayesian framework to estimate model parameters. We illustrate our approach using data from a wild population of blue tits (Cyanistes caeruleus) and present the first estimate of heritability of adult survival in the wild. In agreement with the prediction that selection should deplete additive genetic variance in fitness, we found that survival had low heritability. Because the detection process is incorporated, capture–recapture animal models (CRAM) provide unbiased quantitative genetics analyses of longitudinal data collected in the wild.     

The vascular and cardioprotective effects of liriodenine in ischemia–reperfusion injury via NO-dependent pathway

Liriodenine is an aporphine derivative isolated from the plant Fissistigma glaucescens. Electrophysiological action, particularly the blockage of Na+ and K+ channels, contributes to the drug's well-known anti-arrhythmic action. However, liriodenine's cardioprotective efficacy and the relation of the channel blockages to the efficacy are poorly known, as is the drug's effect on coronary flow and endothelial function. The present study evaluated the protection conveyed by liriodenine to myocardium and coronary endothelial cells under conditions of ischemia-reperfusion and to assess the involvement of a nitric oxide (NO)-dependent mechanism. In the Langendorff model utilizing Sprague-Dawley rat hearts, the left main coronary artery was occluded for 30 min and reperfusion for 120 min. Liriodenine (1 microM) significantly promoted the recovery of coronary flow and decreased myocardial infarction compared with vehicle-treated hearts. The drug attenuated the reduction of endothelial reactivity and NO release. To simulate the condition that occurs in the ischemic stage, human umbilical vein endothelial cells (HUVEC) were cultured in serum free conditions. Liriodenine showed concentration-dependent effects on cell viability associated with anti-apoptosis under serum-deprivation. Liriodenine prevented eNOS reduction in serum-deprived HUVEC and ischemia-reperfusion hearts. The vascular and cardioprotective effects were reversed by N(G)-nitro-L-arginine methyl ester. Another Na+ and K+ channel blocker with similar activities as liriodenine (quinidine) failed to protect endothelial cells and myocytes. These results demonstrate that liriodenine reduces the extent of cardiovascular injuries under ischemia-reperfusion conditions mainly by preserving the eNOS and the NO production.     

Oxidation of buried cysteines is slow and an insignificant factor in the structural destabilization of staphylococcal nuclease caused by H<Subscript>2</Subscript>O<Subscript>2</Subscript> exposure

Summary. The oxidation of buried cysteine or methionine residues can destroy the enzyme activity of a protein by disrupting structure. Engineering in such an oxidatively triggered switch for enzyme activity would only be useful if the effects of substitution are relatively minor, while the effects of the oxidized side chain upon structure are significant and the oxidation relatively easy. To assess the feasibility of this strategy for controlling enzyme activity, the effects of such substitutions and their oxidation were studied in a well characterized model protein, staphylococcal nuclease. Stability and enzyme activity of the oxidized proteins was assessed and compared to the stability and enzyme activity of the unoxidized proteins. Cysteines were found to be generally well tolerated in buried positions but these mutants were not more destabilized than wild-type when oxidized. This shows that buried cysteines are difficult enough to oxidize that this is not likely to be a useful protein engineering strategy or a commonly used regulatory modification. Similar effects were observed for methionine.     

Effect of surfactants on PAH biodegradation by a bacterial consortium and on the dynamics of the bacterial community during the process

The aim of this work was to evaluate the effect of a non-biodegradable (Tergitol NP-10) and a biodegradable (Tween-80) surfactant on growth, degradation rate and microbial dynamics of a polycyclic aromatic hydrocarbon (PAHs) degrading consortium (C2PL05) from a petroleum polluted soil, applying cultivable and non cultivable techniques. Growth and degradation rate were significantly lower with Tergitol NP-10 than that with Tween-80. Toxicity did not show any significant reduction with Tergitol NP-10 whereas with Tween-80 toxicity was almost depleted (30%) after 40 days. Regarding to the cultured bacteria, Pseudomonas and Stenotrophomonas groups were dominant during PAH degradation with Tergitol NP-10, whereas Enterobacter and Stenotrophomonas were dominant with Tween-80. DGGE analyses (PRIMER and MDS) showed that bacteria composition was more similar between treatments when PAHs were consumed than when PAHs concentration was still high. Community changes between treatments were a consequence of Pseudomonas sp., Sphingomonas sp., Sphingobium sp. and Agromonas sp.     

Spatio‐temporal scaling of biodiversity and the species–time relationship in a stream fish assemblage

1. The increase of species richness with the area of the habitat sampled, that is the species–area relationship, and its temporal analogue, the species–time relationship (STR), are among the few general laws in ecology with strong conservation implications. However, these two scale‐dependent phenomena have rarely been considered together in biodiversity assessment, especially in freshwater systems. 2. We examined how the spatial scale of sampling influences STRs for a Central‐European stream fish assemblage (second‐order Bernecei stream, Hungary) using field survey data in two simulation‐based experiments. 3. In experiment one, we examined how increasing the number of channel units, such as riffles and pools (13 altogether), and the number of field surveys involved in the analyses (12 sampling occasions during 3 years), influence species richness. Complete nested curves were constructed to quantify how many species one observes in the community on average for a given number of sampling occasions at a given spatial scale. 4. In experiment two, we examined STRs for the Bernecei fish assemblage from a landscape perspective. Here, we evaluated a 10‐year reach level data set (2000–09) for the Bernecei stream and its recipient watercourse (third‐order Kemence stream) to complement results on experiment one and to explore the mechanisms behind the observed patterns in more detail. 5. Experiment one indicated the strong influence of the spatial scale of sampling on the accumulation of species richness, although time clearly had an additional effect. The simulation methodology advocated here helped to estimate the number of species in a diverse combination of spatial and temporal scale and, therefore, to determine how different scale combinations influence sampling sufficiency. 6. Experiment two revealed differences in STRs between the upstream (Bernecei) and downstream (Kemence) sites, with steeper curves for the downstream site. Equations of STR curves were within the range observed in other studies, predominantly from terrestrial systems. Assemblage composition data suggested that extinction–colonisation dynamics of rare, non‐resident (i.e. satellite) species influenced patterns in STRs. 7. Our results highlight that the determination of species richness can benefit from the joint consideration of spatial and temporal scales in biodiversity inventory surveys. Additionally, we reveal how our randomisation‐based methodology may help to quantify the scale dependency of diversity components (α, β, γ) in both space and time, which have critical importance in the applied context.     

Co‐variation between the intensity of behavioural manipulation and parasite development time in an acanthocephalan–amphipod system

Pomphorhynchus laevis, a fish acanthocephalan parasite, manipulates the behaviour of its gammarid intermediate host to increase its trophic transmission to the definitive host. However, the intensity of behavioural manipulation is variable between individual gammarids and between parasite populations. To elucidate causes of this variability, we compared the level of phototaxis alteration induced by different parasite sibships from one population, using experimental infections of Gammarus pulex by P. laevis. We used a naive gammarid population, and we carried out our experiments in two steps, during spring and winter. Moreover, we also investigated co‐variation between phototaxis (at different stages of infection, ‘young’ and ‘old cystacanth stage’) and two other fitness‐related traits, infectivity and development time. Three main parameters could explain the parasite intra‐population variation in behavioural manipulation. The genetic variation, suggested by the differences between parasite families, was lower than the variation owing to an (unidentified) environmental factor. Moreover, a correlation was found between development rate and the intensity of behavioural change, the fastest growing parasites being unable to induce rapid phototaxis reversal. This suggests that parasites cannot optimize at the same time these two important parameters of their fitness, and this could explain a part of the variation observed in the wild.     

An increase in ethylene sensitivity following pollination is the initial event triggering an increase in ethylene production and enhanced senescence of Phalaenopsis orchid flowers

Cut Phalaenopsis (Phalaenopsis hybrid, cv. Herbert Hager) flowers usually last about 2 weeks. Following pollination however, there is a rapid acceleration of the wilting process, which is completed within 2 days. The first event detected following pollination was an increase in ethylene sensitivity. This increased sensitivity began about 4 h after pollination and peaked 6 h later. A subsequent increase in ethylene production could only be detected 12 to 14 h after pollination. Treatment of the flowers with silver thiosulfate or 1-methylcyclopropene, both inhibitors of ethylene action, completely inhibited the pollination-induced increase in ethylene production and the enhanced senescence of the flowers. This indicates that the pollination-induced increase in ethylene production is a response to the existing ethylene. Treatment of flowers with calcium and its ionophore A23187, which increased ethylene sensitivity and protein phosphorylation, also promoted ethylene production and senescence of unpollinated flowers, EGTA, a calcium chelator, decreased the sensitivity of pollinated flowers to ethylene and delayed and decreased the pollination-induced increase in ethylene production. We suggest that the pollination-induced increase in ethylene sensitivity is the initial pollination-induced event triggering the enhancement of ethylene production, which leads to enhanced senescence of Phalaenopsis flowers.